In previous papers, we showed the porcine gonadotrope population to be composed of three GtH subpopulations that can be separated by density using a continuous Percoll density gradient. We also demonstrated that these subpopulations exhibited different hormonal storage patterns and morphological features during porcine postnatal development at three representative ages: neonates (30 days), prepubers (5-6 months) and matures (16-18 months). In this work, we investigated whether these morphologically heterogeneous subpopulations are also functionally different. Thus, the effect of the hypothalamic gonadotropic hormone-releasing factor (GnRH) on these subpopulations was assessed in order to ascertain whether a mutual relationship between the reported morphological features, hormonal storage patterns and physiological response to the stimulation can be established. For this purpose, gonadotropin secretion was measured by cell immunoblot assay and hormonal content by scanning cytophotometry. Low-density gonadotropes (1.049 g/cm3), present in the three age groups studied, were mainly composed of bihormonal LH/FSH cells in neonates and monohormonal LH cells in prepubers and matures. GnRH stimulation was found to increase both LH and FSH secretion, as well as the intracellular content. These results indicate that GnRH can stimulate both the synthesis and release of both gonadotropins in this subpopulation. Middle-density gonadotropes (1.062 g/cm3), present in prepubers and matures only, were composed of bihormonal cells. GnRH stimulated the secretion of LH and FSH in prepubers and matures, but decreased hormonal contents except that of LH in prepubers. However, GnRH stimulation increased the proportion of immunoreactive gonadotropes (particularly monohormonal cells). Finally, high-density cells (1.087 g/cm3), present in neonates and prepubers only, were mostly composed of bihormonal LH/FSH gonadotropes, and exhibited low (neonates) or no response (prepubers) in terms of LH release and content when treated with GnRH. In conclusion, these results indicate that porcine gonadotrope subpopulations are morphologically and physiologically heterogeneous. The heterogeneity remained through porcine postnatal development, thus suggesting that all the subpopulations are physiologically relevant. However, the different hormonal storage patterns between subsets of the same density suggest age-related differences within each subpopulation due, at least in part, to the different physiological condition of the animals during development.