Use of a yeast-lactobacillus differential microbiological assay permitted investigation into the synthesis of biotin vitamers by a variety of bacteria. A major portion of the biotin activity was found extracellularly. The level of total biotin (assayable with yeast) greatly exceeded the level of true biotin (assayed with lactobacillus). Values for intracellular biotin generally showed good agreement between the assays, suggesting the presence of only true biotin within the cells. Bioautographic analysis of the medium after growth of each organism revealed the presence of large amounts of a vitamer which corresponded to dl-desthiobiotin on the basis of Rf value and biological activity. Biotin, when detected at all, was at very low concentrations. Also, an avidin-uncombinable vitamer was synthesized by a majority of the bacteria. Addition of d-biotin to the growth medium prevented completely the synthesis of both vitamers of biotin. d-Biotin-d-sulfoxide had no effect on the synthesis of desthiobiotin or the avidin-uncombinable vitamer. Addition of dl-desthiobiotin did not prevent its own synthesis nor that of the other vitamer. Control of vitamer synthesis is therefore highly specific for d-biotin. The avidin-uncombinable vitamer was produced only at repressed levels in the presence of high concentrations of both d-biotin and dl-desthiobiotin, which suggested that it is not a degradation product of these substances. A possible mechanism for the overproduction of the biosynthetic precursors of biotin is presented.