The neuroendocrine system plays a key role in the regulation of the secretion of the thymic peptide, thymulin, but it remains to be determined whether thymulin exerts reciprocal regulatory actions on the functional activity of the hypothalamo-pituitary axis. In the present study, we have used a well established in vitro preparation to examine the influence of thymulin on cyclic nucleotide formation and hormone secretion by the rat anterior pituitary gland. Thymulin-Zn<sup>2+</sup>(0.5–50 p M) stimulated the release of immmunoreactive corticotrophin (ir-ACTH), producing effects which were maximal at 10 p M (p < 0.01). At the two highest concentrations tested (10 and 50 p M), italso produced small but significant increases in immunoreactive luteinising hormone (ir-LH) release (p < 0.05), but the secretion of immunoreaffive growth hormone (ir-GH) was unaffected by the peptide (p > 0.05) while that of immunoreactive prolactin (ir-PRL) was reduced (p < 0.01). The ACTH responses to thymulin were accompanied by increased cyclic nucleotide formation. Thus, thymulin (0.5–50 p M) raised the cyclic 3′,5′-adenosine monophosphate (cyclic AMP) content of the pituitary tissue (p < 0.01). At high concentrations (10–50 p M), it also increased cyclic 3′,5′-guanosine monophosphate (cyclic GMP; p < 0.01) accumulation, although lower concentrations of the peptide were ineffective in this regard. The increases in ir-ACTH release provoked by thymulin-Zn<sup>2+</sup>(0.5–5.0 p M) were potentiated markedly by rolipram (1µ M; p < 0.01), a selective inhibitor of the cyclic-AMP-specific phosphodiesterase enzyme. By contrast, zaprinast (10µ M), a selective inhibitor of cyclic-GMP-specific phosphodiesterase, attenuated the corticotrophic responses to higher concentrations of the peptide (10 and 50 p M;p < 0.05). Neither rolipram (1µ M) nor zaprinast (10µ M) influenced the release of ir-LH, ir-PRL or ir-GH in the presence or absence of thymulin-Zn<sup>2+</sup>(0.5–50 p M; p > 0.05). The results suggest that thymulin modulates the secretion of ACTH and possibly LH by the anterior pituitary gland and that its actions are associated with increased cyclic nucleotide formation; in addition, it appears to exert an inhibitory influence on ir-PRL release.