An improved method of high performance liquid chromatography (HPLC) has been developed for the separation and quantitation of low levels of free fatty acids as they occur in mammalian tissues. The fatty acid analysis is based on the esterification of the carboxylic group with 9-anthryldiazomethane (ADAM). HPLC separation and fluorescence measurement of fatty acid ADAM esters allow the determination of pmole amounts of fatty acids. The amounts of free fatty acids of resident and activated alveolar macrophages were determined by the fluorometric HPLC method. There were approximately 2 micrograms/10(6) cells of free fatty acids. In resident macrophages, free 20:4 was a minor component (0.8% of total free fatty acids), while significant amounts of 20:4 were found in the total glycerophospholipids, representing 16.6% of the total fatty acids. A marked increase in amounts of 20:4 (8 times) occurred in activated macrophages stimulated for 1 hr with opsonized zymosan. Small but significant increases (1.5 times) also occurred in other fatty acids. These results show that the release reaction of fatty acids was not selective for 20:4 in alveolar macrophages after the challenge with opsonized zymosan.