The complete alignment of the 63 residues of the mitochondrial ATPase inhibitor from the yeast Candida utilis has been determined. The sequence study was carried out mainly by automatic (liquid and solid-phase) methods. Peptides were obtained by enzymatic digestion with clostripain and purified by reverse-phase high-performance liquid chromatography. The ATPase inhibitor contains three sets of repetitive sequences and eight clusters of charged residues, as also found in the inhibitor of Saccharomyces cerevisiae, with which it shares 58.7% homology of conserved residues. When the two yeast ATPase inhibitor sequences were compared to that of beef heart, 20 residues remained common to the three alignments, although the latter protein contained a long histidine-rich insertion, only found in this inhibitor. Most of the homologous residues were clustered near the center of the protein, which by partial proteolytic digestion of the beef heart ATPase inhibitor [Dianoux, A.C. et al. (1982) FEBS Lett. 140, 223-228] has already been shown to be involved in the biological function.