The ATPase inhibitor protein of the rat liver mitochondrial ATP synthase/ATPase complex has been cloned from a rat liver cDNA library, and its nucleotide sequence determined. The sequence is highly homologous to both the bovine heart (approximately 70%) and the yeast inhibitor proteins (approximately 40%). The deduced protein sequence is 107 amino acids in length, and based on homology to the bovine heart protein, the first 25 N-terminal amino acids encode a putative mitochondrial targeting sequence. The "mature" protein (without the targeting sequence) fused to the maltose binding protein has been overexpressed in Escherichia coli. The maltose binding protein was used as a handle for the development of a rapid one-step purification of the fusion protein by affinity chromatography on an amylose resin. The purified fusion protein was cleaved with Factor Xa protease at the fusion junction, and the resulting ATPase inhibitor protein was purified to > 90% purity. The purified, overexpressed inhibitor protein displays normal inhibitor activity. The protein inhibits ATP hydrolysis catalyzed by the ATP synthase/ATPase complex in submitochondrial particles in a manner kinetically indistinguishable from the same protein purified from rat liver mitochondria, and exhibits a specific activity of approximately 10,000 units/mg. The secondary structure of the inhibitor protein was determined by circular dichroism spectropolarimetry. The experimentally determined structure shows a high content of alpha-helix and is in good agreement with sequence-based structural predictions. As the function of the inhibitor protein is known to exhibit a high dependence on pH, a study of the pH dependence of inhibitor secondary structure was performed. It is shown that as pH is lowered, conditions which activate inhibitory capacity, the protein loses significant alpha-helical structure. This is the first report of the overexpression in E. coli of a functional ATPase inhibitor protein. Secondary structural analysis of this protein indicates that conversion from its active to its inactive form involves a significant conformational change.