We demonstrate that mitogen-activated lymphocytes release a biologically active form of ACTH that stimulates the in vitrorelease of corticosterone from cocultured rat adrenal cells. Neither nonstimulated lymphocytes nor the addition of mitogens alone to adrenal cell cultures had an effect. The steroidogenic activity could be neutralized by rabbit anti-ACTH serum, but not by a nonimmune serum. Both Concana-valin-A- and lipopolysaccharide-stimulated lymphocytes secrete an ACTH-like molecule with an antigenic specificity identical to pituitary-derived ACTH. Further, the amount of measurable immunoreactive ACTH was far lower than the amount of exogenously added ACTH required to evoke such a vigorous glucocorticoid response, suggesting that local deposition of the hormone results in a higher effective ACTH concentration. In addition, lymphocytes physically isolated from adrenal cells by a semipermeable membrane could stimulate steroidogenesis by 48 h, which corresponds to the rise in ACTH detected by RIA. These results confirm that activated lymphocytes synthesize as well as release biologically active ACTH, thus providing an in vitro model for a bidirectional communication between the endocrine and immune systems. © 1993 by The Endocrine Society.