The thymosins are a family of hormone-like products of epithelial cells of the thymus which are important in maintenance and function of the immune system. Thymosin fraction 5, a partially purified extract of calf thymus, can influence pituitary hormone release. We have studied the effects of thymosin α1 (Tα1), the first peptide isolated from thymosin fraction 5, on thyrotropin (TSH), adrenocorticotropin (ACTH), prolactin (Prl) and growth hormone (GH) release. To evaluate its effect in vivo we injected the peptide into the third ventricle of conscious male rats and measured the concentration of the pituitary hormones in plasma at different times after the injection. Following third-ventricular injection of Tα1, there was a significant decrease in plasma TSH and ACTH concentrations in comparison with values of control groups injected with diluent. The decrease in plasma TSH was of longer duration and was obtained with a lower dose of Tα1 than that of ACTH. Also, a significant decrease in plasma Prl was observed, with the same dose as for TSH. On the other hand, there were no significant changes in plasma GH. To examine if there is any direct effect of Tα1 at the pituitary level, we incubated hemipituitaries from male rats in vitro with different concentrations of the peptide. In this system Tα1 evoked a dose-dependent release of TSH and ACTH, while there was no effect on the release of Prl and GH. There was a stimulation of luteinizing hormone (LH) but not follicle-stimulating hormone (FSH) release with a minimal effective dose of 10–12M and a dose-related increase in release to a maximal 7-fold increase in LH at the highest dose (10–7M) evaluated. The results indicate a hypothalamic effect of Tα1 to decrease the release of TSH, ACTH and Prl. On the other hand, acting directly at the pituitary level the peptide stimulates the release of TSH as well as ACTH and selectively stimulates the release of LH but not FSH. Both hypothalamic and pituitary sites of action may be of importance for the interaction between the central nervous, endocrine and immu