Clinical and experimental studies have shown the adverse effects of glucocorticoid deficiency/metyrapone treatment on testicular Leydig cell testosterone production. However, molecular mechanisms that underlie the effects of glucocorticoid deficiency on Leydig cell steroidogenesis are not yet determined. Therefore, the present study was designed to assess the mechanism of this phenomenon. Following metyrapone-induced corticosterone deficiency, serum testosterone, and Leydig cell 14C-glucose oxidation were decreased. StAR mRNA and protein levels were significantly increased in Leydig cells of corticosterone-deficient animals. mRNA levels and the specific activities of P(450)scc and 17beta-HSD were decreased by corticosterone deficiency, whereas the activity and mRNA of 3beta-HSD were increased. Simultaneous administration of corticosterone prevented its deficiency-induced changes in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell testosterone production by decreasing the activities of steroidogenic enzymes and their mRNA expression and glucose oxidation.