A number of studies have reported that a high-fat diet induces increases in mitochondrial fatty acid oxidation enzymes in muscle. In contrast, in two recent studies raising plasma free fatty acids (FFA) resulted in a decrease in mitochondria. In this work, we reevaluated the effects of raising FFA on muscle mitochondrial biogenesis and capacity for fat oxidation. Rats were fed a high-fat diet and given daily injections of heparin to raise FFA. This treatment induced an increase in mitochondrial biogenesis in muscle, as evidenced by increases in mitochondrial enzymes of the fatty acid oxidation pathway, citrate cycle, and respiratory chain, with an increase in the capacity to oxidize fat, as well as an increase in mitochondrial DNA copy number. Raising FFA also resulted in an increase in binding of peroxisome proliferator-activated receptor (PPAR) delta to the PPAR response element on the carnitine palmitoyltransferase 1 promoter. We interpret our results as evidence that raising FFA induces an increase in mitochondrial biogenesis in muscle by activating PPARdelta.