Plasma pseudocholinesterase and porcine liver esterase were used to catalyse the incorporation of the stable isotope oxygen-18 into the carboxyl moiety of lipoxygenase metabolites of arachidonic acid. This simple method produces eicosanoid products containing two oxygen-18 atoms; but the enzymes studied were found to display large substrate specificity in the efficiencies at which oxygen-18 could be incorporated into the lipoxygenase metabolites. Furthermore, [18O2]LTB4 was found not to back exchange during in vitro incubation with human neutrophils. The methods involved for stable isotope incorporation are simple, efficient and produce highly enriched species in a short time. By varying the type of esterase, the amount of esterase or the length of incubation highly enriched species of all eicosanoids tested could be prepared.