Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic mature enzyme when rat liver total RNA was translated in a cell-free system. When the in vitro translation products were incubated with isolated rat liver mitochondria, pre-sulfite oxidase was recovered in mitochondria and converted to the size of the mature enzyme. The in vitro-processed mature enzyme was recovered in the intermembrane space of mitochondria (Ono, H. & Ito, A. (1981) Biochem. Biophys. Res. Commun. 107, 258-264). Mature sulfite oxidase was not imported into mitochondria, and did not affect the import of pre-sulfite oxidase. When mitochondria were incubated with gel-filtered translation products, the import was dependent on ATP, and the activity restored by the addition of ATP was blocked by valinomycin and K+ ion. These results suggest that the import of pre-sulfite oxidase into mitochondrial intermembrane space requires an electrochemical potential across the inner membrane. When mitochondria were fractionated, most of the processing activity was recovered in the mitoplast, whereas the inner membrane (after being mostly inverted by sonication) exhibited only slight activity. The processing activity was strongly inhibited by some metal chelators including EDTA, GTP, and Zincon. It was not inhibited by phenyl methyl sulfonyl fluoride, aprotinin, or various microbial protease inhibitors including pepstatin, antipain, leupeptin, and chymostatin. The processing enzyme seems to be a metal protease. The processing of pre-sulfite oxidase by mitoplasts was energy-dependent.