The action of 25-hydroxy-6,19-dihydro-6,19-epidioxyvitamin D3 [25-(OH)D3 endoperoxides, 2a and 3a] in inducing differentiation of human myeloid leukemia cells (HL-60) was studied by using their radioactive derivatives (2a' and 3a'). When HL-60 cells were incubated with the labeled endoperoxides (2a' and 3a') in serum-free RPMI 1640 medium, no radioactivity was incorporated into either the cytosol or the chromatin fraction of the cells. When the radioactive endoperoxide (2a') was incubated in the culture medium for 3 days, with or without HL-60 cells, about 45% of the compound was similarly converted to 19,25-dihydroxy-6,19-dihydro-6,19-epoxyvitamin D3 (4a) and about 10% to 25-hydroxy-6,19-epoxyvitamin D3 (6a). These two new vitamin D derivatives were synthesized chemically and tested for their biological activities. Both compounds (4a and 6a) were about 2 times as active as 25-(OH)D3 endoperoxides (2a and 3a) and about 7 times as active as 25-hydroxyvitamin D3 (1a) in inducing differentiation of HL-60 cells. The differentiating activity of these compounds was well correlated with their activity in binding to the cytosol receptor for 1 alpha, 25-dihydroxyvitamin D3 in HL-60 cells. The in vitro bone-resorbing activity of 25-hydroxy-6,19-epoxyvitamin D3 (6a) and 25-(OH)D3 endoperoxide (2a) was higher than that of 25-hydroxyvitamin D3 (1a), indicating that the differentiating activity also paralleled the bone-resorbing activity in these vitamin D derivatives. These results suggest that 25-(OH)D3 endoperoxides (2a and 3a) induce differentiation of HL-60 cells and bone resorption after being converted to these two compounds.