3-Methylcrotonyl-CoA carboxylase has been purified to near homogeneity from maize leaves. The resulting preparations of 3-methylcrotonyl-CoA carboxylase have a specific activity of between 200 and 600 nmol.min-1.mg-1 protein, representing an approximately 5000-fold purification of the enzyme. The purified 3-methylcrotonyl-CoA carboxylase has a molecular weight of 853,000 +/- 34,000 and is composed of two types of subunits, a biotin-containing subunit of 80 +/- 2 kDa and a non-biotin-containing subunit of 58.5 +/- 1.5 kDa. These data suggest that the enzyme has an alpha 6 beta 6 configuration. The optimum pH for activity is 8.0. The kinetic constants for the substrates 3-methylcrotonyl-CoA, ATP, and HCO3- are 11 microM, 20 microM, and 0.8 mM, respectively. Kinetic studies of the 3-methylcrotonyl-CoA carboxylase reaction with variable concentrations of two substrates confirmed that ATP and HCO3- bind sequentially to the enzyme and that ATP and 3-methylcrotonyl-CoA bind in ping-pong fashion. However, similar analyses indicate that the binding of HCO3- at the first site is affected by 3-methylcrotonyl-CoA. Kinetic studies of the role of Mg2+ in the 3-methylcrotonyl-CoA carboxylase reaction establish that Mg.ATP is the substrate for the enzyme, that free ATP is an inhibitor, and that free Mg2+ is an activator. Both Mn2+ and Co2+ can substitute somewhat for Mg2+, but Zn2+ is unable to do so. In addition to carboxylating 3-methylcrotonyl-CoA, the maize carboxylase can carboxylate crotonyl-CoA, but not acetoacetyl-CoA. In fact, acetoacetyl-CoA is a potent, noncompetitive inhibitor, which indicates that the enzyme contains an acetoacetyl-CoA binding site that is independent of the active sites. The monovalent cations K+, Cs+, Rb+, and NH4+ activated 3-methylcrotonyl-CoA carboxylase activity, with Rb+ being the most potent activator. The inhibition of 3-methylcrotonyl-CoA carboxylase by sulfhydryl and arginyl modifying reagents could be partly alleviated by the substrates ATP and 3-methylcrotonyl-CoA, which suggests that sulfhydryl and arginyl residues may be involved in catalysis.