Two variant clones of a human myeloid leukemia cell line (HL-60) resistant to the active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], were isolated from a 1 alpha,25(OH)2D3-sensitive parent clone, and the mechanism of the resistance was examined. When the parent clone was incubated with 120 nM 1 alpha,25(OH)2D3, cell growth was suppressed to half of the control and about half of the cells exhibited phagocytic activity and C3 rosette formation on day 3. The variant clones, however, were resistant to 120 nM 1 alpha,25(OH)2D3. One of the variant clones was also insensitive to other potential inducers such as 12-O-tetradecanoyl-phorbol-13-acetate, actinomycin D, and dimethyl sulfoxide. When the variant clones were incubated with 1 alpha,25(OH)2[3H]D3, they took up much less radioactivity than the parent clone into the whole cells, and into the cytosol protein-bound and chromatin-bound fractions. The variant clones were found to possess reduced amounts of the cytosol receptor protein to which 1 alpha,25(OH)2D3 was specifically bound; but the hormone-receptor complex could be transferred to the chromatin acceptor sites similarly both in the wild type clone and its variant clones, indicating that one of the major defects in the 1 alpha,25(OH)2D3-resistant clones is the reduced amounts of the specific cytosol receptor. These results support the concept that 1 alpha,25(OH)2D3 induces differentiation of human myeloid leukemia cells by a receptor-mediated mechanism.