The total cytosol activity of acetyl-CoA carboxylase (acetyl-CoA:CO(2) ligase (ADP), EC 18.104.22.168) in the liver is known to be 6- to 10-fold higher in genetically obese hyperglycemic mice (C57BL/6J-ob) than in nonobese mice. The results of immunochemical titrations, Ouchterlony double-diffusion analysis, and kinetic and heat inactivation studies indicated that this rise in the level of carboxylase activity in liver extracts from obese mice was ascribed to an increase in the quantity of the enzyme protein, which was indistinguishable from that derived from nonobese mice. Combined immunochemical and isotopic techniques showed that the rate of synthesis of the carboxylase per liver was 7.7-fold higher in obese than in nonobese mice. The rate of degradation of the carboxylase was found to be 1.7-fold lower in obese than in nonobese mice, the half-life being 115 and 67 hr, respectively. These results indicate that the increase in the acetyl-CoA carboxylase content of the liver in obese mice is due mainly to a rise in the rate of enzyme synthesis, and in a minor degree, to a decrease in the rate of enzyme degradation.