Antibodies were raised against the beef heart mitochondrial ATPase inhibitor. This antiserum prevented the ability of the ATPase inhibitor to inhibit the F1 ATPase activity. Peptide fragments obtained by enzymatic cleavage of the inhibitor protein were tested by immunoblotting or ELISA for their response to the anti-inhibitor antiserum. An antigenic determinant was located in the sequence spanning His 48 to Lys 58 of the inhibitor molecule.