Steroidogenesis was investigated in Leydig cell-enriched fractions isolated from the testes of rats dosed dermally with 130 mg/kg/day hexafluoroacetone (HFA) for 14 days and from pair-fed control rats. Compared to controls, Leydig cells from HFA-treated rats exhibited decreased incorporation of [14C]acetate (78%) and [3H]mevalonate (41%) into sterols and steroids. Testosterone was decreased 50% in the testes of HFA-treated rats. Incubation of Leydig cells from untreated rats with 1.0 mM HFA did not affect steroidogenesis. HFA treatment led to the development of histopathological lesions in the testes within 24 hr after a single dose; with daily dosing the lesions became progressively more severe. Despite the development of severe lesions, HFA treatment did not affect the blood levels of luteinizing hormone or testosterone; however, follicle-stimulating hormone was slightly elevated (48%) after 14 days of treatment. The data indicate that steroidogenesis is inhibited in Leydig cells of HFA-treated rats; the inhibition is not due to a direct or immediate effect of HFA, nor does it appear to be hormonally mediated.