Actithiazic acid, an antibiotic known to interfere with the biotin metabolism of certain bacteria, did not significantly increase or decrease the degree of biotin deficiency (measured by liver and muscle total biotin content) induced in the rat by administration of raw egg white. Two biotin-like substances, “avidin uncombinable biotin” (AUB) and SC factor, have been found in the supernatant of Escherichia coli cultures as well as in the urine and stool of the rat. The “avidin uncombinable biotin” of the urine of actithiazic acid-treated rats was increased about ten times over the concentration of AUB found in the urine of untreated controls. The AUB of the stool was similarly but less consistently increased. The AUB and SC factors are normally produced in small quantities by E. coli, but under the influence of actithiazic acid this production is increased five-to tenfold. This increase of biotin vitamers was found in supernatants of cultures of crude rat stool flora and in pure cultures of a coliform organism (isolated from this rat stool) as well as in similar cultures of a pure strain of E. coli K-12. These findings suggest an enteric origin of AUB and SC. Moreover, no AUB could be detected in rat muscle or liver, as contrasted with the large amounts found in stool and urine. No significant amount of “true” biotin is elaborated into the medium by E. coli, although the organisms themselves contain considerable biotin in bound form. AUB and SC have been separated chromatographically, but their chemical identification has not been accomplished.