Abstract: A respiration-deficient nuclear mutant of Saccharomyces cerevisiae was isolated that lacked mitochondrial adenosine triphosphatase (F1). The absence of this enzyme was established by adenosine triphosphatase assays, by antibody-binding studies, and by radioimmunochemical tests for F1 subunits. The mutant also lacked mitochondrial 32Pi-ATP exchange activity and exhibited greatly reduced amounts of cytochromes aa3, b, and c1. It possessed a mitochondrial protein-synthesizing system, but several products of this system were either abnormal or missing. Thus, the level of the three mitochondrially synthesized subunits of cytochrome c oxidase was at least 10 times lower than in the wild type. The mutant was unable to grow anaerobically even if the growth medium contained a fermentable substrate and unsaturated lipids. This property has not yet been reported for any S. cerevisiae strain. The mutant had a great tendency to lose its mitochondrial genome. The conversion to the double mutant state decreased the growth rate by a factor of 20 to 30. These results suggest that mitochondrial adenosine triphosphatase has an essential function even in nonrespiring cells. The mutant described here offers new possibilities for studying the assembly of F1 from its subunits and the role of F1 in mitochondrial biogenesis.