Biotin-requiring mutants have been isolated fromEscherichia coliK-12 and classified into four nutritional groups on the basis of their responses to various biotin vitamers, the ability to feed other mutant strains, and the nature of vitamers excreted into the growth medium. The growth response of the mutant strains to various biotin vitamers was in a linear sequence starting from 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid, to desthiobiotin, and to biotin. From the results of the cross-feeding experiments the excretion of three types of vitamers was indicated and two of the vitamers were identified as desthiobiotin and 7-keto-8-aminopelargonic acid. The third vitamer could not be detected by bio-autographic technique. From these results a biosynthetic pathway of biotin inE.colihas been proposed.