Cyclosporine induces hypoandrogenism in adult male rats. In order to assess whether this effect of CsA may be due to a direct inhibitory effect on Leydig cell function, CsA (0, 50, 500, and 5000 ng/ml) was added to a collagenase-dispersed mixed Leydig cell preparation and incubated with and without hCG (0, 0.1, 0.3, 1.0, 3.0, and 10.0 ng/ml). Testosterone (T) production, mitochondrial cholesterol side chain cleavage (CSCC) and microsomal 17,20-desmolase enzyme activities in Leydig cells were determined after 3 hr of incubation. In the absence of CsA, stimulation of T production was maximal (about 16-fold) with 1.0 ng/ml hCG. With 50 and 500 ng/ml CsA there were no changes in either the hCG-stimulated T levels or the two enzymatic activities. However, 5000 ng/ml CsA significantly (P less than 0.05) reduced the hCG (1 ng/ml)-stimulated T levels, CSCC and 17,20-desmolase activities. The high dosage of CsA (5000 ng/ml) also caused a significant decrease in cell viability (P less than 0.05) during the incubation period. These effects of CsA were not due to cremophor EL, the CsA vehicle. This in vitro data indicate that high dosages of CsA (greater than or equal to 5000 ng/ml) appear to have a cytotoxic effect on rat Leydig cells that results in a decrease in T production. However, lower doses of CsA (less than 500 ng/ml) do not have any direct inhibitory effect on the rat Leydig cells, suggesting that the hypoandrogenic effect of in vivo CsA in rats is not due to any direct effect on the testis.