The vitamin D3 derivatives, 1alpha-hydroxyvitamin D3; 1alpha-hydroxy-3-deoxyvitamin D3; 5,6-trans-vitamin D3; and 5,6-trans-25-hydroxyvitamin D3 were tested for bone resorbing activity in vitro. 1alpha-Hydroxy-3-deoxyvitamin D3 and 5,6-trans-vitamin D3 were inactive at concentrations as high as 10(-6)M. 1alpha-Hydroxyvitamin D3 had significant effects on mineral and matrix resorption at concentrations of 2.5 X 10(-8)M and above, and was 2-3 orders of magnitude less potent than 1,25-dihydroxyvitamin D3 in this system. A concentration of 2.5 X 10(-7)M of 5,6-trans-25-hydroxyvitamin D3 was required to stimulate 45Ca release. The results indicate that 1) in the vitamin D3 series of compounds the presence of only one hydroxyl group in the molecule in either the 1alpha or the 3beta position in the A ring results in a compound with very limited or no direct effects on bone, and 2) the direct effects of vitamin D3 congeners on bone resorption do not require the presence of a 25-hydroxy group, but their activity is markedly enhanced by the 25-hydroxy group.