A functional interaction between testicular macrophages and Leydig cells has been suggested. The present study attempts to clarify the interaction between purified Leydig cells and macrophages from adult male rats in coculture, employing culture conditions that maintain Leydig cell steroidogenic responsiveness in vitro. Basal Leydig cell testosterone production over 24 h was not significantly affected by coculture with macrophages, but an inhibitory effect of testicular macrophages on testosterone production by Leydig cells over 24 h was observed in the presence of increasing doses of LH from 0.125 ng/ml up to a maximally stimulating dose of 8 ng/ml. A consistent inhibitory effect was observed over a range of Leydig cell-testicular macrophage coculture ratios from 0.5:1 to 4:1 in the presence of LH (8 ng/ml). A similar inhibitory effect on maximal LH-stimulated Leydig cell testosterone production over 24 h was observed when Leydig cells were cocultured with peritoneal macrophages. Conditioned medium collected from testicular or peritoneal macrophage cultured for 24 h also inhibited LH-stimulated Leydig cell testosterone production, indicating that the effect of the macrophages was mediated by a secreted product. Inhibition of LH-stimulated testosterone production was observed also when Leydig cells were cultured in the presence of testicular macrophages for 24 h before maximal LH stimulation (8 ng/ml) for a further 24 h. Human recombinant interleukin-la and interleukin-1β (0.5-10 U/ml) did not significantly alter basal or LH-stimulated Leydig cell testosterone production at 24, 48, or 72 h of culture. The specific binding of125I-human CG to Leydig cells was not affected by testicular macrophage-conditioned medium. These data demonstrate that testicular and peritoneal macrophages inhibit LH-stimulated Leydig cell testosterone production in coculture through secreted factors, acting distal to the LH receptor, and provide further support for paracrine interactions between these cell types.