In addition to reconstituting immune competence, the thymus gland preparation, thymosin fraction 5 (TSN-5), has recently been shown to stimulate secretion of hormones from the hypothalamic-pituitary adrenal axis in vivo and from pituitary corticotropes in vitro. The purpose of the present study was to investigate the effects of TSN-5 on secretion of immunoreactive @-endorphin (i@-E) by mouse corticotropic tumor cells. The release of i@-E by AtT-20 pituitary tumor cells was increased in a dose-dependent manner by concentrations of 30-600 pg/ml of TSN-5, whereas concentrations greater than 1,000 pglml were increasingly less effective in stimulating secretion. TSN-5 (600 pg/ ml) significantly stimulated iP-E release within 7 min; maximal secretory responses (up to 275% of control release) occurred by 4 hr. The secretory response of AtT-20 cells to 600 pg/ml TSN-5 (37.9 k 2.0 vs. 16.1 k 1.0 ng i@-E/ml/4 hr, mean f SE) was similar in magnitude to release evoked by 0.1 pM corticotropin-releasing factor (CRF). Combining TSN-5 and CRF treatments increased secretion of i@-E to nearly 600% of control levels, an effect greater than an additive influence of the two independent treatments. Whereas CRF treatment reduced the levels of i@-E in AtT-20 cell extracts after 24-hr treatment by 45% (231.8 24.7 vs. 417.2 k 17.8 ng i@-Elmg protein, CRF vs. vehicle treatments, respectively), TSN-5 did not significantly alter cellular hormone content. Neither TSN-al nor TSN-04, two of the component peptides of TSN-5, affected basal or CRF-stimulated release of 10-E, indicating that an unidentified constituent(s) is corticotropic. This study demonstrates that TSN-5 directly stimulates hormone release from AtT-20 cells and potentiates the secretory actions of CRF. These findings support the concept of interactions between neuroendocrine and immune regulators of the hypothalamic-pituitary adrenal axis. Additionally, corticotropic tumor cells appear to be a useful model in which to differentiate a hormonally active constitutent(s) of TSN-5 and to evaluate the cellular mechanisms of action of TSN-5 on corticotropic cells.