Vitamin D-like steroids added to the culture medium induce a specific calcium-binding protein (CaBP) in embryonic chick duodenum maintained in organ culture. This system provides a biologically relevant assay, i.e., a physiological response in a principle target organ, for the study of the relative biopotency of vitamin D metabolites and analogs. A number of fluoro analogs of vitamin D 3 (D 3) and its metabolites were assayed in the present study. Analogs fluorinated in the lα position (1α-F-D 3) or in both the 1α and 25 positions (1α,25-F 2-D 3) were markedly more potent than vitamin D 3 itself although 1α,25-F 2-D 3 was only 1 7 th as potent as 1α-F-D 3. The 25-fluoro analog (25-F-D 3) was a very weak inducer; only 1 45 th as potent as vitamin D 3. The 25-fluoro analog of 1α-hydroxyvitamin D 3 (1α-OH-25-F-D 3) was less potent than its nonfluorinated counterpart. Although 25-fluorination reduced biopotency in all other analogs tested, 24 R-OH-25-F-D 3 was about 15 times more potent than 24 R,25-(OH) 2-D 3. Of considerable interest was the effect of difluorination at the 24-carbon position: both 24,24-F 2-25-OH-D 3 and 24,24-F 2-1α,25-(OH) 2-D 3 were about four times as potent as their nonfluorinated counterparts. The 24,24-F 2-1α,25-(OH) 2-D 3 is, therefore, the most potent vitamin D 3 analog yet tested in this system i.e., it is four times more potent than the most potent naturally occurring vitamin D 3 metabolite, 1α,25-(OH) 2-D 3.