Exposure of myeloid leukemia cells to analogs of vitamin D results in monocytic-like maturation of several variants of these cells. We report here that brief treatment of HL60 cells with differentiation-inducing concentrations of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) makes these cells resistant to cell death by apoptosis. Resistance was studied by using the calcium inophore A23187 and the cancer chemotherapeutic drugs 1-beta-D-arabinocytosine and etoposide. Apoptosis was detected by the characteristic morphology under light microscopic examination, presence of DNA "ladders" on agarose gel electrophoresis, DNA fragmentation by filter elution assay, and the "apoptotic index" obtained by comparison of damage to mitochondrial and nuclear genes on Southern blots. The protective effect of 1,25(OH)2D3 treatment was apparent before the phenotypic evidence of differentiation and before altered traverse of the cell cycle could be detected. Northern and Western blot analysis showed that the expression of bcl-2 proto-oncogene was rapidly reduced by 1,25(OH)2D3, which excludes the involvement of this gene in the protective effect. The rapidity of the protective effect of 1,25(OH)2D3 is consistent with the hypothesis that the activation of the monocytic differentiation program is sufficient to interfere with programs that lead to cell death by apoptosis.