ABSTRACT. Hypercalcemia and hypercalciuria in sarcoidosis are thought to result from the endogenous overproduction of an active vitamin D metabolite. We employed primary cultures of pulmonary alveolar macrophages from two patients with biopsy-proven pulmonary sarcoidosis and a recent or current clinical abnormality in calcium metabolism to synthesize in vitro a 1,25- dihydroxyvitamin D₃ [l,25-(OH)₂D₃]-like metabolite from 25-hydroxyvitamin D₃ (25OHD)₃. The macrophage metabolite co- chromatographed with [³(H]i,25-(OH)₂D₃ on normal phase and reverse phase high performance liquid chromatography and was bound with high affinity by the chick intestinal receptor for 1,25-(OH)₂D₃. On UV spectroscopy, the metabolite possessed) the carbon-5,7,10 (19) cis-triene chromophore characteristic of a vitamin D sterol. Electron impact mass spectrometry of tri-methylsilyl ether derivatives of the metabolite revealed a mass fragmentation pattern similar to that of the trimethylsilyl ether (derivative of authentic 1,25-(OH)₂D₃. The incubation of cultured) macrophages from two patients with idiopathic pulmonary fibrosis and two with scleroderma with [³H]25OHD)₃ did not result n i production of a metabolite with the chromatographic identity of 1,25-(OH)₂D₃. These data indicate that the metabolite of (25OHD₃ synthesized by sarcoid macrophages in vitro is 1,25-(OH)₂D₃ and that the macrophage is a synthetic source of the sterol metabolite in sarcoidosis.