We investigated the metabolism of 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 (26,27-F6-1,25(OH)2D3, ST-630) and 1 alpha,25-dihydroxyvitamin (1,25(OH)2D3) in cultured neonatal mouse calvaria to elucidate why ST-630 is more potent than 1,25(OH)2D3 in stimulating bone resorption in organ culture. The metabolites were extracted with ethyl acetate or chloroform/methanol (1:1) from cultured calvaria or medium incubated with [3H]-ST-630 or [3H]-1,25(OH)2D3 for various periods, and separated by high performance liquid chromatography. [3H]-ST-630 in cultured calvaria was converted to [3H]-26,26,26,27,27,27-hexafluoro-1 alpha,23(S),25-trihydroxyvitamin D3(26,27-F6-1,23,25(OH)3D3,ST-232), which stimulated bone resorption equipotently as 1,25(OH)2D3. The amount of [3H]-ST-232 produced in the bone increased with passage of the culture period. In contrast, the amount of [3H]-ST-630 in the bones decreased in the 2 day cultures. In the medium, [3H]-ST-630 was hardly detectable for 2 days. This suggests that ST-630 is metabolized to ST-232 which is retained in the bones. On the other hand, some [3H]-1,25(OH)2D3 was metabolized to inactive forms detectable in the medium. Therefore, the high potency of ST-630 in stimulating bone resorption in organ culture may be associated with a difference between ST-630 and 1,25(OH)2D3 in the mode of metabolism in the cultured bones.