New highly active isomers of the natural hormone 1alpha, 25-dihydroxyvitamin D3 possessing an exomethylene group at the 2-position were prepared in a convergent manner, starting with (-)-quinic acid and the corresponding (20R)- and (20S)-25-hydroxy Grundmann ketones. These 2-methylene-19-norvitamins were efficiently converted to the 2-methyl and 2-hydroxymethyl derivatives, some of which exhibited pronounced in vivo biological activity. Configurations of the A-ring substituents were determined by 1H NOE difference spectroscopy as well as by spin decoupling experiments. It was established that the bulky methyl and hydroxymethyl substituents at C-2, due to their large conformational free energies, occupy mainly equatorial positions. Additionally, hydroxylation of the C(10)-C(19) double bond in 1alpha,25-(OH)2D3 was performed, resulting in 1alpha,19,25-trihydroxy-10,19-dihydrovitamin D3 derivatives in which the hydroxymethyl substituent at C-10, for steric reasons, is forced to occupy an axial position. In consequence, the vitamin D3 analogues were synthesized in which the 1alpha-hydroxy group, required for biological activity, is almost exclusively axially or equatorially oriented because of stabilization of the single A-ring chair conformations. The relative ability of the synthesized analogues to bind the porcine intestinal vitamin D receptor was assessed and compared with that of the natural hormone. It was established that vitamins possessing the axial orientation of the 1alpha-hydroxy substituent exhibit a significantly increased receptor binding affinity. Compounds with a 2-methylene substituent showed selective calcemic activity profiles, being extremely effective on bone calcium mobilization. 2alpha-Methyl-substituted vitamins proved to be much more active in vivo than the corresponding epimers with 2beta-configuration. All of the 2-substituted vitamins exhibited pronounced HL-60 differentiating activity, those 2alpha-substituted in the 20S-series being especially potent. The present studies imply that the axial orientation of the 1alpha-hydroxy group is necessary for biological activity of vitamin D compounds.