Ketoconazole is an antifungal agent capable of inhibiting human steroid hormone synthesis, including renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] synthesis. The ability of this drug to inhibit the extrarenal production of 1,25-(OH)2D, as occurs in human granuloma-forming disease states, including sarcoidosis, has not been evaluated. We examined the effect of ketoconazole on the 1,25-(OH)2D-calcium homeostatic mechanism in a hypercalcemic patient with sarcoidosis and on the synthesis of 1,25-(OH)2D3 in vitro by cultured pulmonary alveolar macrophages (PAM) from this and another host. Oral ketoconazole therapy (800 mg/day) decreased the serum 1,25-(OH)2D concentration 73% within 4 days; this was associated with a 15% decrease in the serum calcium concentration and a 57% decrease in the fractional urinary calcium excretion rate. In vitro, ketoconazole had a rapid onset, concentration-dependent inhibitory effect on sarcoid PAM 1,25-(OH)2D3 synthesis (ED50 = 0.1 mumol/L) that was not reversible by exposure to leukotriene C4, a potent stimulator of PAM 1,25-(OH)2D3 synthesis. Kinetic analysis of ketoconazole's action on the macrophage 1 alpha-hydroxylation reaction was examined at concentrations achieved in vivo when the drug is given orally. The velocity of the 1 alpha-hydroxylation reaction at ketoconazole concentrations of 0.01-1.0 mumol/L increased as the concentration of substrate 25-hydroxyvitamin D3 increased from 12-2000 nmol/L.