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Protein kinase C (PKC)-theta, a member of the 'novel' subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for 'classical' PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr(538)), turn motif (Ser(676)) and hydrophobic motif (Ser(695)). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical in vivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor kappaB (NF-kappaB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-kappaB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-kappaB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive phosphorylation of its activation loop.