Two new antimetabolites of biotin were isolated from culture filtrates of Streptomyces lydicus: β-methyldethiobiotin and β-methylbiotin. 14C-biotin or 14C-pimelic acid was not incorporated into either of these antimetabolites by the growing culture. Neither of the compounds could substitute for the biotin requirement in Saccharomyces cerevisiae. Both compounds had a strong and rather specific antimicrobial effect against mycobacteria. Their antimicrobial activities were reversed by biotin. Both compounds had an affinity for avidin.