In this study we evaluated the quantitative influence of GRF and TRH on the rate of hormone secretion from single cells in cultures of male pituitaries. To accomplish this, we dispersed pituitaries from male rats with trypsin and cultured them for 24 or 48 h. Reverse hemolytic plaque assays for GH and prolactin were then performed on retrypsinized cultures to identify individual cells that secreted these hormones. Mammotropes and somatotropes were found to comprise 31.4 ± 1.8 and 32.2 ± 0.9% (mean ± SE, n = 3 experiments), respectively, of all cells in 24-hour cultures. Immunocytochemical staining of different batches of cells from the same dispersions corroborated the proportions of these two cell types. Differences in the rate of basal hormone secretion were observed within each of these cell populations as evidenced by the gradual appearance of prolactin and GH plaques over a 4-hour period when incubations were conducted in the absence of stimulatory secretagogues. Addition of increasing concentrations of GRF (1 × 10–10–1 × 10–7M) orTRH (1 × 10–9–1 × 10–6M) to these incubations resulted in dose-related increases in the rate of GH and prolactin plaque formation, respectively. Maximal plaque development by somatotropes could be induced within 30 min of administering large doses of GRF, indicating that most, if not all somatotropes are responsive to this secretagogue. In contrast, approximately one third of all mammotropes could not be stimulated to form plaques acutely when subjected to similar treatment with TRH. This observation suggests that mammotropes are heterogeneous with respect to TRH responsiveness. Finally, treatment with these secretagogues did not increase over the control value the percentage of all cultured cells that formed plaques, demonstrating that the hypothalamic factors tested did not recruit additional cells into the secretory pool. These results provide evidence to indicate that: (1) mammotropes may account for a greater proportion of pituitary cells in males than previously believed; (2) mammotrope and somatotrope populations in male rats are each heterogeneous with respect to basal hormone secretion, and that mammotropes differ in responsiveness to TRH, and (3) the rate of plaque formation provides a reliable index of the rate of hormone secretion. Thus, the reverse hemolytic plaque assay provides a semiquantitative tool for studying hormone release by individual pituitary cells in culture.