Bismuth is present in several popular over-the-counter drugs for nausea and diarrhea and is occasionally abused by patients with chronic gastrointestinal disorders. The most common consequence of bismuth overdose is neurological dysfunction. In experimental animals, bismuth overdose results in lowered serum testosterone levels, suggesting that reproductive dysfunction may be an additional component of bismuth toxicity. Although the precise mechanisms responsible for the lowered testosterone levels are unknown, it has been shown that bismuth accumulates within testicular macrophages. This may be important because these cells, which are commonly found in direct contact with Leydig cells, are known to exert paracrine influences on the Leydig cells for local control of testosterone production. However, bismuth may also exert direct effects on Leydig cells because it passes by these cells on its way to the phagocytic macrophages. The purpose of the present studies was to isolate both testicular macrophages and Leydig cells from rat testis and study the direct effects of bismuth on these cells with regard to their viability and function. We found that when Leydig cells were treated for 24 h with bismuth (1-100 microM) no change in viability or secretion of testosterone was observed. However, when testicular macrophages were similarly treated with bismuth a significant effect on viability was observed with as little as 6.25 microM bismuth, with near-complete cell death at 50 microM after 24 h. However, bismuth had no effect on the viability on testicular macrophages at 50 microM up to 8 h, therefore, we studied the secretion of tumor necrosis factor alpha (TNF-alpha) after 4 h of exposure to 50 microM bismuth and found no influence on the production of TNF-alpha. Taken together, it seems likely that bismuth has no direct effects on Leydig cells but, rather, lowers testosterone levels by killing testicular macrophages, thereby interrupting their local paracrine influence on Leydig cells through factors other than TNF-alpha.