In Escherichia coli, degradation of abnormal proteins is an energy-requiring process; it is decreased in mutants in the lon (capR or deg) gene. We find that the protein encoded by the lon gene is an ATP-dependent protease and is identical to protease La, recently described in E. coli. Both proteins are serine proteases that hydrolyze casein and globin, but not insulin, in the presence of ATP and Mg2+. Both respond to ATP, less well to other nucleoside triphosphates, and not to nonhydrolyzable ATP analogs. The purified lon protein has an apparent Mr of 450,000 and appears to be composed of four identical subunits. Its size, chromatographic behavior, and sensitivity to various inhibitors and heat are indistinguishable from those of protease La. Moreover, in a strain that carries additional copies of the lon+ allele on a plasmid, the content of protease La, but not of other proteases, is 2- to 10-fold greater than in the lon+ parent strain. Strains carrying the nonsense mutations capR9 and capR- also contain this ATP-dependent proteolytic activity, but it is present in substantially lower amounts and is inactivated by phosphocellulose chromatography, unlike the wild-type enzyme. Degradation of abnormal proteins in these lon- strains, which is slower than in the wild type, still requires ATP. Alterations in the ATP-dependent protease in the lon- mutants can account for the defect in intracellular proteolysis and perhaps also for the other phenotypic effects of this pleiotropic gene.