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.