L-Phenylalanine was found to be a competitive inhibitor of pyruvate kinase in the rat prostate, seminal vesicles, uterus, and skeletal muscle. In contrast, L-alanine exerted no appreciable effect on pyruvate kinase activity but was capable of protecting against, as well as reversing, the inhibition produced by L-phenylalanine. The results suggest that the interactions between L-phenylalanine and L-alanine are important to the regulation of pyruvate kinase activity in tissues containing the M-type isozyme.