The relative capacities for testicular macrophages and resident peritoneal macrophages to secrete the pro-inflammatory cytokine, interleukin 1 (LL-1), in response to stimulation by bacterial lipopolysaccharide (LPS), were compared in vitro. Macrophages were isolated from adult male rat testicular interstitial cells or peritoneal lavage by adherence to glass coverslips or plastic culture dishes. The macrophages were immediately cultured, with or without a maximal dose of LPS (1 mu g/ml), over 24 hours at 32 degrees Celsius. Bioactive LL-1 production was measured by a sensitive thymocyte proliferation bioassay, employing recombinant human LL-1 beta as the reference standard. In comparison with the peritoneal macrophages, testicular macrophages displayed only a very small response to LPS, producing 2.8% of the amount of LL-1 per cell secreted by peritoneal macrophages cultured under identical conditions. Production of authentic LL-1 was confirmed by inhibition of the bioassay response in the presence of human recombinant LL-1 receptor antagonist. A small molecular mass (<10 kDa based on ultrafiltration) inhibitor of LL-1 bioactivity was also present in the medium collected from both cultures, but this inhibitory activity did not account for the differences in activity observed. In cultures of total peritoneal cells under similar conditions, addition of testosterone (10-1,000 ng/ml) did not affect LL-1 production in response to LPS. These data indicate that testicular macrophages have a reduced ability to secrete bioactive IL-1, and they provide further evidence for an altered capacity for immune responses within the testis.