The subcellular localization of enzymes involved in leukotriene formation was analysed according to biological (chemotaxis, spasmogenic properties) and analytical methods. By subcellular fractionation the major activity for 5--lipoxygenase and L-gamma-glutamyltranspeptidase coeluted with the 200,000 g precipitate while glutathione-S-transferase activity was mainly present in the 200,000 g supernatant. Our data were supported by results indicating that the 200,000 g precipitate and supernatant fractions proved to be most active in generating 5-HETE and leukotriene C4 (LTC4) respectively. The 200,000 g pellet was the most active fraction in transforming synthetic LTC4 into LTD4 and LTE4. When synthetic LTD4 was incubated with the various subcellular fractions and the appearance of LTE4 was analysed the 20,000 and 200,000 g pellets were the most potent fractions. Several discrepancies observed using biological, biochemical and analytical (synthetic substrates) methods may be in part due to the formation of leukotriene isomers which interfere with the biological assays.