We evaluated the biological activity of two sets of ring A stereoisomers of 2-methyl-1α,25-dihydroxyvitamin D 3 (2-methyl-1α,25(OH) 2D 3) and 2-methyl-20-epi-1α,25-dihydroxyvitamin D 3 (2-methyl-20-epi-1α,25(OH) 2D 3) in terms of the following: transactivation of a rat 25-hydroxyvitamin D 3-24-hydroxylase gene promoter including two vitamin D response elements (VDREs) and a human osteocalcin gene promoter including a VDRE in transfected human osteosarcoma (MG-63) cells; a vitamin D receptor (VDR)-mediated response using a VDR–GAL4 one-hybrid luciferase reporter system and a retinoid X receptor α (RXRα)-mediated response using an expressed VDR/RXRα–GAL4 modified two-hybrid luciferase reporter system in transfected human epitheloid carcinoma, cervix (HeLa) cells; and modulation of cell surface CD11b antigen expression in human leukemia (HL-60) cells. All the diastereomers of both analogues exhibited unique biological activity profiles depending upon the configurations of the C-1 and C-3 hydroxyl groups, the C-2 methyl group in ring A, and the C-20 methyl group in the side chain. Of the eight possible diastereomers of the 2-methyl analogues, 2α-methyl-1α,25(OH) 2D 3 was the most potent and exhibited comparable or even greater biological potency than 1α,25(OH) 2D 3. Of the eight possible diastereomers of the 2-methyl-20-epi analogues, 2α-methyl-20-epi-1α,25(OH) 2D 3 was the most potent and exhibited 100- to 200-fold higher transcriptional potencies than 1α,25(OH) 2D 3 and exceptionally high cell regulatory activities. 2β-Methyl-20-epi-1α,25(OH) 2D 3 was nearly as potent as its 2-epimer, 2α-methyl-20-epi-1α,25(OH) 2D 3, whereas its 20-epimer, 2β-methyl-1α,25(OH) 2D 3, was almost completely biologically inactive. In these respects, it can be postulated that the double modification of 2-methyl substitution and 20-epimerization to 1α,25(OH) 2D 3 induces remarkable changes in a VDR/RXRα/VDRE-mediated signaling response and greatly enhances biological activity. The other striking finding was that 2β-methyl-20-epi-3-epi-1β,25(OH) 2D 3 is transcriptionally more active than 1α,25(OH) 2D 3 despite lacking the 1α-hydroxyl group, which was believed to be essential for expressing VDR-mediated gene transcription. Since the C-20 natural counterpart, 2β-methyl-3-epi-1β,25(OH) 2D 3, was almost completely biologically inactive, 20-epimerization is probably responsible for activation of gene expression. Although earlier extensive structure–activity studies of vitamin D analogues showed stereochemistry at the C-1, C-3, and C-20 of 1α,25(OH) 2D 3 to be the key structural motif for vitamin D action, our results clearly demonstrated that stereochemistry at the C-2 is also an important structural motif for vitamin D action and imply that 2-methyl substitution possibly induces conformational changes in ring A depending upon the combinations of configurations of the C-1 and C-3 hydroxyl groups with C-20 stereochemistry. Consequently, several of these analogues exhibit exceptionally high or unexpected biological activities at the molecular and cellular levels. These results suggest that 2-methyl substitution together with alterations of stereochemistry in both ring A and the side chain of 1α,25(OH) 2D 3 will provide useful analogues for structure–activity studies and development of therapeutic agents with unique biological activity profiles.