Lipoxin A₄ (LXA₄) possesses potent bioactions. To facilitate its detection, an enzyme‐linked immunosorbent assay (ELISA) was developed that proved sensitive and selective. Quantitation by ELISA of LXA₄ generated from cellular sources strongly correlated (r=0.99) with values obtained by high‐pressure liquid chromatography (HPLC). We used this LXA₄‐ELISA to examine parameters influencing LXA₄ generation from endogenous substrates during human platelet‐neutrophil (PLT‐PMN) interactionsin vitro. Agonist‐induced LXA₄ production was clearly evident at a PLT‐PMN ratio of 10∶1, and recombinant human granulocyte/monocyte colony stimulating factor‐priming of PMN augmented LXA₄ generation 5–6 fold. The chemotactic peptide formylmethionyl‐leucyl‐phenylalanine, platelet‐derived growth factor and arachidonic acid (20∶4n−6) each stimulated formation of immunoreactive LXA₄ (iLXA₄) in these co‐incubations. The presence of iLXA₄ was also evaluatedin vivo in aspirin‐sensitive asthmatic patients who, in a randomized, double‐blind crossover design, underwent nasal lavage after they each ingested a predetermined threshold dose of aspirin or placebo. Aspirin challenge provoked statistically significant increases in iLXA₄ in each patient (P<0.005). These results validate the use of a solid‐phase ELISA for detection of LXA₄. Furthermore, the use of this ELISA has allowed the first documentation of iLXA₄ formation in human subjects with aspirin‐sensitive asthma following specific antigenic challenge.