Mitochondrial aspartate aminotransferase, an enzyme localized on the inner face of the inner mitochondrial membrane, is released into the intermembrane space upon addition of a "movement effector" (succinate, fumarate, pyruvate, or glutamate) [Waksman, A., & Rendon, A. (1974) Biochimie 56, 907-924]. After removal of the movement effector, 90% of the released enzyme rebound to mitoplasts. Lubrol fractionation showed that this bound activity was associated with the inner membrane. Internalization was demonstrated by using both enzymatic and molecular approaches. It was found that 70% of the reassociated enzyme became inaccessible from the outside of the mitoplast either to a nonpermeating substrate (NADH), to mild protease hydrolysis, or to recognition by a specific antibody. In contrast, in inside-out vesicles, the enzyme remained accessible to NADH, protease, and antibodies. Latency measurements performed at different temperatures on whole intact mitochondria confirmed the existence of reversible intermembrane movement of the enzyme in situ.