The transmembrane glycoprotein CD36 has been identified in isolated cell studies as a putative transporter of long-chain fatty acids. To examine the physiological role of CD36, we studied FA uptake and metabolism by tissues of CD36 null mice after injection with two fatty acid analogs. Compared to controls, uptake was substantially reduced (50–80%) in heart, skeletal muscle, and adipose tissues of null mice. The reduction in uptake was associated with a large decrease in fatty acid incorporation into triglycerides, which could be accounted for by an accumulation of diacylglycerides. Thus CD36 facilitates a major fraction of fatty acid uptake by myocardial, skeletal muscle, and adipose tissues, where it is highly expressed. Its role in other tissues where its expression is low and cell-specific could not be determined in these studies.