Studies into the effects of oligornerization on FOF,ATPsynthase function are contradictory. We optimized the in-gel ATPase assay to investigate the functional differences of monomers versus dimers. In Triton X-100 extracts of heavy bovine heart mitochondria (HBHM) and mitoplasts, but not submitochondrial particles (MgATP-SMP), dimers had greater specific activity than monomers: at 30 degrees C, the dimer/monomer activity ratios were 2.3, 1.4, and 1.0, respectively. These differences in HBHM and mitoplasts extracts were enhanced at 37 degrees C but lost at 20 degrees C. In mitoplasts but not in MgATP-SMP, dimers were selectively shielded from limited chymotrypsin degradation of F, a subunit, possibly due to interactions with other proteins or ligands in the native inner membrane. Despite these differences, all three preparations had similar percentages of dimers and similar contents of the native inhibitor IF, in Vm (monomer) and (dimer) Vd. These results suggest that, in native membrane, monomers and dimers are functionally distinct.