The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, and the fluorescence emission intensity of mitochondrial F1-ATPase was examined. Purified F1 from bovine heart mitochondria was slowly inactivated at low denaturant concentration, and inactivation was associated with delta and epsilon subunit dissociation. delta and epsilon subunits were bound together to form a stable and soluble heterodimer. In parallel, appearance of turbidity was observed. This was caused by the formation of alpha3beta3gamma non-covalent aggregates, as analyzed by SDS-PAGE. Short periods of exposition of the F1 complex to high concentrations of guanidine hydrochloride (0.8-3 M) again induced deltaepsilon dissociation as a heterodimer and the formation of an inactive alpha3beta3gamma subcomplex. This eventually dissociated progressively into single subunits caused by partial unfolding, as evidenced through changes of the protein intrinsic fluorescence emission. Our results suggest that the delta and epsilon subunits are loosely bound to alpha3beta3gamma , and play an important role in determining structural stability to isolated mitochondrial F1-ATPase.