Despite the significance of mitochondrial ATP synthase for mammalian metabolism, the regulation of the amount of ATP synthase in mammalian systems is not understood. As brown adipose tissue mitochondria contain very low amounts of ATP synthase, relative to respiratory chain components, they constitute a physiological system that allows for examination of the control of ATP synthase assembly. To examine the role of the expression of the P1-isoform of the c-F_o subunit in the biogenesis of ATP synthase, we made transgenic mice that express the P1-c subunit isoform under the promoter of the brown adipose tissue-specific protein UCP1. In the resulting UCP1p1 transgenic mice, total P1-c subunit mRNA levels were increased; mRNA levels of other F₁F_o-ATPase subunits were unchanged. In isolated brown-fat mitochondria, protein levels of the total c-F_o subunit were increased. Remarkably, protein levels of ATP synthase subunits that are part of the F₁-ATPase complex were also increased, as was the entire Complex V. Increased ATPase and ATP synthase activities demonstrated an increased functional activity of the F₁F_o-ATPase. Thus, the levels of the c-F_o subunit P1-isoform are crucial for defining the final content of the ATP synthase in brown adipose tissue. The level of c-F_o subunit may be a determining factor for F₁F_o-ATPase assembly in all higher eukaryotes.—Kramarova, T. V., Shabalina, I. G., Andersson, U., Westerberg, R., Carlberg, I., Houstek, J., Nedergaard, J., Cannon, B. Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-Fo subunit P1 isoform.