The formulation of media for selective, automatable, radiometric detection of growth of Mycobacterium tuberculosis in vitro is described. Palmitic-1 acid labeled with carbon-14 and formic-14C acid were compared as substrate sources of [14C]O2 in media deficient in carbohydrate and containing appropriate antimicrobial agents that are not active against tubercle bacilli. A preliminary clinical laboratory study of a medium containing 4 microCi palmitic-1-14C acid per ml showed that this method might provide the basis for practical laboratory use.