The time course of biotin synthesis and the distribution of the vitamin were studied in Escherichia coli growing in biotin-free and biotin-supplemented media. In biotin-free media, biotin synthesis occurred at a maximum rate just prior to the onset of the logarithmic phase of growth with continuous excretion of the vitamin into the growth medium. The bound biotin levels of the cells varied depending on cultural conditions, but the content of intracellular free biotin was always the highest at an early incubation time. At the end of the logarithmic phase the majority of 'total' biotin synthesized was found extracellularly. However, the extent of overproduction of 'true' biotin, particularly in an enriched medium, was very small. In biotin-supplemented media, the time course of synthesis was completely different and the total amount of biotin synthesized varied inversely with the concentration of (+)-biotin present prior to the inoculation of bacteria . The inhibition of biotin synthesis was achieved only by (+)-biotin; other biotin vitamers had no effect. The results suggest that biotin synthesis is controlled by (+)-biotin. (Author).