Saccharomyces cerevisiae pet mutants, of complementation group G115, are deficient in mitochondrial ATPase and have properties indicative of defective F1. In this study we show that C287/LU1, a mutant belonging to group G115, is complemented by the yeast nuclear ATP3 gene coding for the gamma-subunit of the mitochondrial F1-ATPase. The amino-terminal sequence of the mature gamma-subunit matches the sequence encoded by ATP3 starting with the 34th amino acid confirming the identity of the gene, and earlier evidence indicating that this F1 component is synthesized as a precursor with a long amino-terminal extension. The properties of the mitochondrial ATPase have been studied in C287/LU1 with an Ala273-->Val substitution in the carboxyl-terminal region of the gamma-subunit and in W303 delta ATP3, a mutant lacking the gamma-subunit as a result of a deletion in ATP3. Both strains have negligible ATPase activity but near normal concentrations of the alpha- and beta-subunits of F1. In W303 delta ATP3, the subunits do not form a stable F1 oligomer nor are they firmly associated with F0. This is not true of C287/LU1, which was found to assemble an F1-F0 complex. These data indicate that the yeast gamma-subunit has dual functions, one in catalysis of ATP hydrolysis/synthesis and the second in assembly/stability of F1.