Salmonella typhimurium is able to synthesize cobalamin (B12) under anaerobic growth conditions. The previously described cobalamin biosynthetic mutations (phenotypic classes CobI, CobII, and CobIII) map in three operons located near the his locus (minute 41). A new class of mutant (CobIV) defective in B12 biosynthesis was isolated and characterized. These mutations map between the cysB and trp loci (minute 34) and define a new genetic locus, cobA. The anaerobic phenotype of cobA mutants suggests an early block in corrin ring formation; mutants failed to synthesize cobalamin de novo but did so when the corrin ring is provided as cobyric acid dicyanide or as cobinamide dicyanide. Under aerobic conditions, cobA mutants were unable to convert either cobyric acid dicyanide or cobinamide dicyanide to cobalamin but could use adenosylcobyric acid or adenosylcobinamide as a precursor; this suggests that the mutants are unable to adenosylate exogenous corrinoids. To explain the anaerobic CobI phenotype of a cobA mutant, we propose that the cobA gene product catalyzes adenosylation of an early intermediate in the de novo B12 pathway and also adenosylates exogenous corrinoids. Under anaerobic conditions, a substitute function, known to be encoded in the main Cob operons, is induced; this substitute function can adenosylate exogenous cobyric acid and cobinamide but not the early biosynthetic intermediate. The cobA gene of S. typhimurium appears to be functionally equivalent to the btuR gene of Escherichia coli.