Much progress has been made in the identification of proteins that mediate mitochondrial fusion, as well as in the elucidation of their role in mitochondrial quality control. Yet, while deficiency of mitochondrial fusion seems to induce a mitochondrial dysfunction suggesting a requirement of fusion for oxidative metabolism, the cause of the dysfunction is unclear. We have undertaken a bioenergetics analysis to understand the basis of the mitochondrial dysfunction, using intact mouse embryonic fibroblasts deficient in mitofusin1 and mitofusin2 (DKO) incubated under conditions replete in glycolytic vs. oxidative substrate. We first noted an approximate doubling of the nonmitochondrial oxygen consumption (JO2) in DKO cells compared to wildtype (Wt) MEFs, regardless of substrate. Upon switching from primarily glycolytic to more oxidative conditions, DKO MEFs, similar to the Wt, increased basal mitochondrial JO2 (oligomycin-sensitiveþoligolycin-insensitive, non-mitochondrial subtracted), although the rise was smaller in DKO cells (an increase of 55511 vs. 114511 pmoles/min/40k cells in DKO vs. Wt; values: mean5sem). When only the oligomycin-sensitive respiration is considered, the rise was similar in DKO cells (an increase of 121.3510 vs. 135.8513 pmoles O 2 /min/40k cells in DKO vs. Wt), whereas the oligomycin-sensitive JO2 decreased in DKO cells only (by~50%). In both Wt and DKO cells, the rate of media acidification declined upon switching from glycolytic to oxidative substrate, indicating a shift from glycolytic to oxidative ATP production. Thus, as in Wt MEFs, DKO MEFs demonstrate a basal mitochondrial respiration controlled by ATP turnover and substrate oxidation. Differently from the Wt, DKO mitochondria can exert a significant control of basal JO2 by proton leak, which could function to compensate for any limitation in mitochondrial capacity.