Previous reports have demonstrated that introduction of fluorine atoms at C-26 and C-27 of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) results in the potentiation of various aspects of some biological activities. The higher biological activities of 26,26,26,27,27,27-hexafluoro- 1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) were accounted for in part by a decrease in metabolic inactivation via the 26- and 27-hydroxylation pathways. In addition to 26,27-F6-1,25-(OH)2D3 not being hydroxylated in the 26 and 27 positions, it did not undergo 24-hydroxylation despite a significant induction by 26,27-F6-1,25-(OH)2D3 of 24-hydroxylase activity in the HL-60 cell system. Another fluorinated vitamin D3 analog, 26,26,26,27,27,27-hexafluoro-1 alpha-hydroxyvitamin D3 (26,27-F6-1 alpha-OH-D3) may not undergo 25-hydroxylation as efficiently as 1 alpha-OH-D3 in vivo because a rise in serum 26,27-F6-1,25-(OH)2D3 levels after injection of 26,27-F6-1 alpha-OH-D3 was delayed significantly with a much smaller amplitude. Furthermore, 26,26,26,27,27,27-hexafluoro-1,23(S),25-trihydroxyvitamin D3 retained full activity in the induction of HL-60 cell differentiation even after 23(S)-hydroxylation, in contrast to 1,23(S),25-(OH)3D3. These data suggested that substitution of fluorines for hydrogens at C-26 and at C-27 positions may result in alteration in chemical reactivity and/or conformation of C-23, C-24 and C-25 positions of the 1,25-(OH)2D3 molecule.