A series of analogs of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] with multiple fluorine substitutions in positions 26 and 27 have been tested for their activities 1) in competing with 1,25-(OH)2D3 for binding to partially purified chick intestinal cytosol, 2) in stimulating resorption of fetal rat limb bones in vitro and 3) in competing with 25-hydroxyvitamin D3 for binding sites in rat plasma. The relative potencies of 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D3, 26,26,26-trifluoro-25-hydroxyvitamin D3, 27-nor-26,26,26-trifluoro-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 in competing for intestinal cytosolic binding were 17:11:1:1. The relative potencies of the same series of compounds on stimulating resorption of fetal rat bones was 25:21:9:1. The relative ability of these four compounds to compete for plasma binding sites was 0.3:0.3:0.4:1.0. The results indicate that multiple fluorine substitution in the vicinity of the 25-hydroxyl group can markedly enhance the direct effects of 25-hydroxyvitamin D3 on its target tissues. This is postulated to result from the electronegativity of the fluorines which increases the acidity of the 25-hydroxyl group and enhances its affinity for the receptor. In contrast to the effects seen with the 25-hydroxyvitamin D3 analogs, multiple fluorine substitution in positions 26 and 27 did not enhance the activity of 1,25-(OH)2D3 on either cytosolic binding or bone resorption. Presumably, this is because biological activity, expressed in terms of affinity for the receptor, is already optimal in the 1,25-(OH)2D3 structure. The relative activities of the 26,27-hexafluoro derivative and 1,25-(OH)2D3 in competition for the plasma binding sites was 0.4:1.0.(ABSTRACT TRUNCATED AT 250 WORDS)