In non-lipogenic tissues malonyl-CoA serves two functions: 1) regulation of mitochondrial fatty acid oxidation via inhibition of carnitine palmitoyltransferase-I (CPT-I), and 2) providing two-carbon units for fatty acid chain elongation. In the presence of acetyl-CoA carboxylase (ACC-2) substrates acetyl-CoA, ATP-Mg2+, and bicarbonate isolated rat heart mitochondria synthesize malonyl-CoA that results in inhibition of CPT-I. Furthermore, addition of palmitoyl-CoA and NADPH/NADH to isolated intact rat heart mitochondria catalyze the incorporation of radioactive malonyl-CoA into lipid soluble products suggesting an outer mitochondrial membrane localization of the long-chain fatty acid elongation pathway. These data suggest that malonyl-CoA formed by mitochondrial ACC-2 is available for both, regulation of fatty acid oxidation and for fatty acid chain elongation. The finding with perfused heart that stable isotope labeled palmitate is chain-elongated to stearate without prior chain shortening by β-oxidation and isotope dilution as judged from the near identical isotope enrichment of palmitoyl- and stearoylcarnitine is in support of this. These data and the exclusive outer mitochondrial membrane localization of CPT-I suggests that mitochondria catalyze long-chain fatty acid elongation also in vivo. Our data raise the question about metabolic channeling of malonyl-CoA between ACC-2, CPT-I, and fatty acid chain elongation and how these processes are regulated. Supported by: DK-066107, POI AG15885, and VA Med. Res. Serv.