Abstract: The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F(1) ATPase (or assembly intermediates thereof). Mutants lacking the alpha or beta subunits of F(1), or the Atp11p and Atp12p chaperones that promote F(1) assembly, have normal levels of the bicistronic ATP8/ATP6 mRNAs but fail to synthesize Atp6p and Atp8p. F(1) mutants are also unable to express ARG8(m) when this normally nuclear gene is substituted for ATP6 or ATP8 in mitochondrial DNA. Translational activation by F(1) is also supported by the ability of ATP22, an Atp6p-specific translation factor, to restore Atp6p and to a lesser degree Atp8p synthesis in the absence of F(1). These results establish a mechanism by which expression of ATP6 and ATP8 is translationally regulated by F(1) to achieve a balanced output of two compartmentally separated sets of ATP synthase genes.