Pro-opiomelanocortin (POMC) gene expression and POMC peptides have been demonstrated in the Leydig cells of the testis, although selective removal of the Leydig cells with the cytotoxic drug ethane dimethane sulfonate did not significantly reduce levels of testicular POMC mRNA or peptides in adult rats. Since macrophages in the rat spleen synthesize POMC peptides, we investigated whether isolated macrophages from the adult rat testis may be an additional source of POMC-derived peptides. Testicular macrophages were isolated by collagenase treatment of adult rat testes and adherence to siliconized glass coverslips; the biological, cytochemical and immunological characteristics of the attached cells were compared with those of Leydig cells purified by Percoll gradient centrifugation. Macrophages in the cell preparations were identified by positive esterase cytochemical staining, latex bead ingestion, and immunocytochemical staining with ED2 (a macrophage-specific monoclonal antibody), and an absence of 3 beta-hydroxysteroid dehydrogenase cytochemical staining. Leydig cells in the purified preparations were positive for 3 beta-hydroxysteroid dehydrogenase and esterase staining but negative with ED2, and were not phagocytic. Based on these criteria, the purities of the macrophage and Leydig cell preparations employed in this study were estimated to be 87 +/- 4% and 91 +/- 3%, respectively. Cytoplasmic beta-endorphin (beta EP) immunoreactivity (ir) was present in 62 +/- 9% of cells in the purified Leydig cell preparations--confirming these cells as a source of POMC-derived peptides. In addition, ir-beta EP and ir-ACTH were localized to the cytoplasm of a similar proportion of cells (beta EP, 62.5 +/- 5%; ACTH, 64 +/- 5%) in macrophage preparations.(ABSTRACT TRUNCATED AT 250 WORDS)