We present evidence which demonstrates that L-cycloserine, structural analog of L-alanine, which is known to be an effective aminotransferase inhibitor, is also a potent inhibitor of cellular pyruvate metabolism. This effect was found to be related to its almost instantaneous action in decreasing pyruvate concentrations in a dose-dependent manner. 1H nuclear magnetic resonance studies clearly demonstrate that the irreversible removal of pyruvate induced by L-cycloserine is caused by the decarboxylating action of the latter. Pyruvate disappearance induced by L-cycloserine can be stoichiometrically accounted for as acetate. The process does not involve any chemically detected transformation of L-cycloserine. These observations lead to two main considerations regarding the known action of L-cycloserine. First, its inhibitory effect on gluconeogenesis from lactate could be explained only on the basis of its ability to reduce pyruvate availability with no apparent need for transaminase inhibition. Second, its ability as a transaminase inhibitor should be reconsidered in view of its potent decarboxylating action on pyruvate and probably other oxoacids.