While it has been shown that culture medium from testicular macrophages can influence testosterone production when added to Leydig cells, the identity of the factor(s) responsible for this activity remains unknown. Since tumor necrosis factor alpha (TNF alpha) has been shown to be capable of influencing testosterone production by Leydig cells, a series of studies was conducted to determine if testicular macrophages produce TNF alpha. It was found that testicular macrophages from adult rats produce a factor that is capable of lysing L929 cells, which are used as a traditional bioassay for TNF alpha. The TNF alpha activity in the macrophage-conditioned medium could be neutralized by the addition of anti-murine TNF alpha but not with the addition of preimmune IgG. While lipopolysaccharide (LPS) slightly increased the release of TNF alpha, neither follicle-stimulating hormone (FSH) nor testosterone had a similar effect. It was not determined if the isolation procedure had artificially 'activated' the macrophages. Medium from cultured Sertoli cells, Leydig cells and peritubular cells did not contain TNF alpha activity. These studies are consistent with the hypothesis that the paracrine interaction between testicular macrophages and Leydig cells is mediated in part by TNF alpha.