Scatchard analyses of 1,25-dihydroxyvitamin D receptors (VDR) from chick and rat intestine, bovine thymus, pig kidney cells (LLC-PK1), and human breast cancer cells (T-47D) demonstrated that 1,25-dihydroxyvitamin D3 (1,25-D3) and 1,25-dihydroxyvitamin D2 (1,25-D2) had equal affinities for VDR. 24-Epi-1,25-dihydroxyvitamin D2 (24-epi-1,25-D2) exhibited affinities for VDR equal to that of 1,25-D2 and 1,25-D3 in most of these tissues. Scatchard analysis with 24-epi-[3H]1,25-D2 underestimated total VDR by 50-70% in rat intestine, LLC-PK1, and T-47D cells. The biological activity of 24-epi-1,25-D2 was found to be only 30-70% of 1,25-D3 and 1,25-D2 as determined by in vivo induction of intestinal calcium transport and bone calcium resorption in the rat and in vitro induction of 23- and 24-hydroxylase activities in T-47D cells. In vivo tracer kinetic studies demonstrated that in the rat 1,25-D3 and 1,25-D2 kinetics were similar, whereas 24-epi-1,25-D2 had a 25% shorter plasma half-life and was cleared from the body 2.8 times faster than the natural hormones. This more rapid clearance of 24-epi-1,25-D2 along with reduced VDR binding appears to explain the reduced biological activity of 24-epi-1,25-D2. Our data clearly demonstrate that although there are differences in side chain structure between 1,25-D2 and 1,25-D3, the VDR binding, biological activity, and whole body tracer kinetics of these two metabolites are virtually identical. However, movement of the 28 methyl of 1,25-D2 from its natural S configuration to the R configuration significantly alters the activity of this hormone.