The product of the lon (capR or deg) gene in Escherichia coli is protease La, an ATP-dependent protease with a linked ATPase activity. Unlike most lon mutations, capR9 is dominant over the wild type under certain conditions. When protease La was isolated from R9 cells and from a recessive capR- strain using DEAE-cellulose chromatography, the mutant enzymes showed about 50% of the wild type activity. Unlike the wild type, the R9 and R- proteases were inhibited by addition of NaCl (less than 0.1 M). In addition, the R9, but not the R-, material inhibited protelysis by normal protease La, and this effect may account for its dominant phenotype. When isolated by phosphocellulose chromatography, the R9 protein lost proteolytic activity but still inhibited the wild type enzyme. This inhibitory activity was purified to near homogeneity using DEAE-cellulose and heparin-agarose chromatography, and corresponded to the 94,000-dalton R9 gene product. At different concentrations, it inhibited ATP-dependent casein degradation and casein-stimulated ATP hydrolysis to a similar extent. Thus, rates of ATP and protein cleavage remained proportional. Similar inhibition of the wild type protease was observed in the presence of DNA which stimulates both protein and ATP hydrolysis. Half-maximal inhibition was observed with approximately a 1:1 ratio of the R9 to the wild type protein. The subunit sizes of the R9 and the wild type protease were indistinguishable but they differed in isoelectric points. Upon gel filtration, both eluted as tetramers (450,000 daltons) in the absence of salt. However, with 0.1 M NaCl, the wild type protease La remained as a tetramer, but the R9 protein dissociated into dimers and monomers and became a more effective inhibitor. After mixing with R9 protein, 3H-labeled protease La remained tetrameric, though it had lost activity. These findings suggest that tetramer formation between the wild type and defective R9 subunits is responsible for the inhibition of the proteolytic and ATPase activities.