Radiolabeled fatty acid incorporation and measurement of its subsequent release is a standard method of studying the response of the alveolar macrophage to agonist stimulation. However, it has never been verified whether this method provided an accurate reflection of the endogenous fatty acid pool activity. In order to determine the validity of studies performed with radiolabeled fatty acid probes, we directly compared their release with the actual release of the endogenous fatty acids determined using a fluorescent derivatization method. Although we found that significant amounts of arachidonate are released following tannin and glucan exposure, the radiolabeled arachidonate is released much more rapidly than the endogenous arachidonate pool it was taken to represent. In contrast, the endogenous pools of the saturated fatty acids palmitate and stearate are more actively released by glucan than the radiotracer method indicated. These differences arise from inherent limitations of the radiotracer method which are confounded when examining macrophage fatty acid metabolism.