We have previously demonstrated low levels of immunoreactive (ir)-β-endorphin (β-EP) and ir-ACTH in a subpopulation of mouse spleen macrophages, which is consistent with an involvement of opioid peptides in modulation of immune responses. Gel chromatography studies suggested the presence of an ~3.5,000-molecular weight (mol wt) species, putatively β-EP, an ~11.5,000-mol-wt species, putatively β-lipotropin, and a higher molecular weight species (putative β-EP precursor, pro-opiomelanocortin (POMC)). In this study we have extended our original findings by demonstrating the presence of messenger RNA for POMC by the use of a complementary DNA probe and Northern blot analysis of extracts of mouse and rat spleen. In addition, using high performance liquid chromatography (HPLC), we have shown that the major endorphin species in mouse spleen macrophages is β-EP1-31, and that there are smaller amounts of each of the acetylated forms, N-acetyl-β-EP1-16 (α-endorphin), N-acetyl-β-EP1-17 (γ-endorphin), N-acetyl-β-EP1-27, and N-acetyl-β-EP1-31. We interpret these studies as showing that (a) the spleen is an organ of POMC synthesis and that (b) the predominant COOH-terminal product of macrophage POMC is the opiate-receptor active species β-EP1-31.