The regulation of large conductance calcium- and voltage-activated potassium (BK) currents by activation of the protein kinase C (PKC) and glucocorticoid signalling pathways was investigated in AtT20 D16:16 clonal mouse anterior pituitary corticotroph cells. Maximal activation of PKC using the phorbol esters, 4β-phorbol 12-myristate, 13-acetate (PMA), phorbol 12, 13 dibutyrate (PDBu) and 12-deoxyphorbol 13-phenylacetate (dPPA) elicited a rapid, and sustained, inhibition of the outward steady-state voltage- and calcium- dependent potassium current predominantly carried through BK channels. The effect of PMA was blocked by the PKC inhibitors bisindolylmaleimide I (BIS; 100 nM) and chelerythrine chloride (CHE; 25 μM) and was not mimicked by the inactive phorbol ester analogue 4α-PMA. PMA had no significant effect on the 1 mM tetraethylammonium (TEA)-insensitive outward current or pharmacologically isolated, high voltage-activated calcium current. PMA had no significant effect on steady-state outward current in cells pre-treated for 2 h with 1 μM of the glucocorticoid agonist dexamethasone. Dexamethasone had no significant effect on steady-state outward current amplitude or sensitivity to 1 mM TEA and did not block PMA-induced translocation of the phorbol ester-sensitive PKC isoforms, PKCα and PKCe, to membrane fractions. Taken together these data suggest that in AtT20 D16:16 corticotroph cells BK channels are important targets for PKC action and that glucocorticoids inhibit PKC signalling downstream of PKC activation. In many neuroendocrine cells of the anterior pituitary gland, activation of the protein kinase C (PKC) intracellular signalling pathway leads to sustained cellular excitability and neurosecretion although the cellular mechanisms and targets for PKC are poorly understood (Ozawa & Sand, 1986; Mason et al. 1988). In anterior pituitary corticotrophs PKC mediates the sustained phase of adrenocorticotrophin (ACTH) secretion stimulated by activation of the phospholipase C pathway by the hypothalamic secretagogue vasopressin (Carvallo & Aguilera, 1989; Oki et al. 1990). Vasopressin elicits a biphasic elevation of intracellular free calcium (Corcuff et al. 1993; Tse & Lee, 1998) and during the sustained phase of calcium influx stimulates PKC translocation and enhances PKC activity at the plasma membrane, an effect that is mimicked by the cell-permeant PKC-activating phorbol esters (Carvallo & Aguilera, 1989). In AtT20 mouse corticotroph cells phorbol-ester-mediated activation of PKC has been proposed to exert effects both distal and proximal to voltage-dependent calcium influx, which may result from activation of different PKC isoforms (McFerran et al. 1995) to elicit ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995). Intracellular free calcium measurements in AtT20 cells suggest that PKC-induced calcium influx results, at least in part, from inhibition of TEA-sensitive potassium conductances. This inhibition results in membrane depolarization and subsequent, indirect, enhancement of voltage-gated calcium influx (Reisine & Guild, 1987; Reisine, 1989). However, ionic conductances regulated by PKC activation in corticotrophs have not been identified. In rat GH4C1 pituitary cells activation of PKC results in inhibition of the TEA-sensitive large conductance calcium- and voltage-activated potassium (BK) channels (Shipston & Armstrong, 1996), which act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997). Furthermore, BK channels are an important target for cellular regulation by two distinct, physiologically relevant, intracellular signalling pathways in AtT20 corticotrophs. Activation of the cAMP-dependent protein kinase pathway results in inhibition of BK channels leading to a robust secretory response. Protein kinase A (PKA)-mediated inhibition of BK channel function is blocked by activation of a protein-synthesis-dependent signalling cascade activated by glucocorticoid hormones. The cross-talk between these two pathways at the level of BK channels is an important determinant of the secretory response in this system (Shipston et al. 1996; Lim et al. 1998; Tian et al. 1998). As TEA-sensitive BK channels act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997) and PKC-mediated calcium influx is dependent upon inhibition of a TEA-sensitive conductance in AtT20 cells (Reisine & Guild, 1987; Reisine, 1989), we have addressed whether BK channels are an important cellular target for PKC action in this system. Furthermore, as glucocorticoids block PKC-stimulated ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995) and antagonize PKA-mediated inhibition of BK channels in this system (Shipston et al. 1996; Tian et al. 1998) we have addressed the question as to whether glucocorticoids also block PKC-mediated regulation of BK channels in AtT20 D16:16 corticotroph cells.