The pro-opiomelanocortin (POMC) gene encodes a family of peptides originally identified in the pituitary gland. An important POMC-derived peptide hormone, corticotropin (ACTH), is also produced by leukocytes and modulates in vitro immune functions. The present investigation was undertaken to determine the kinetics and cellular distribution of ACTH immunoreactivity (ACTH-ir) in vitro in rat splenic leukocyte subpopulations. Cells were cultured with Concanavalin A (ConA), lipopolysaccharide (LPS), or media alone. ACTH-ir was identified with a specific antiserum raised against ACTH 1-24. Double indirect-immunofluorescence was done at 0, 21, and 48 h for B, T-helper (Th), and T-cytotoxic (CTL) cells. Initial kinetic studies demonstrated peak ACTH-ir in all cell types at 18-21 h for both ConA and LPS treatments. A few leukocytes (1-2%) expressed ACTH-ir at 0 h and these were found to be macrophages (MØ). Lymphocyte ACTH-ir is 0% at 0 h and rises to 90 ± 5% and 75 ± 6% at 21 h with ConA and LPS, respectively. This sharply contrasts with 9 ± 4% of each cell type positive in media alone at 21 h. The percent immunoreactivity among the three lymphocyte subpopulations did not significantly differ at any single treatment at a single time point. However, there were significant differences in the intensity levels among the subpopulations. At 48 h of ConA or LPS treatment only 10 ± 4% of B, Th and Tc were positive, while none were positive in media alone. Stimulated peritoneal MØ also increase positivity for ACTH-ir (85 ± 5%). These results indicate that rat splenic B, CTL, and Th lymphocytes can be immunologically stimulated to express the peptide hormone ACTH and that basal ACTH expression in macrophages is distinct from that in lymphocytes. Thus, lymphocyte-derived ACTH may be a paracrine or autocrine regulator of immune function. © 1995.