5-Lipoxygenase pathway-derived products of arachidonic acid released by human eosinophils activated in vitro have been measured by using radioimmunoassays specific for leukotriene B4 (LTB4) and for sulfidopeptide leukotrienes including leukotriene C4 (LTC4). Eosinophil-enriched leukocytes (mean, 85% eosinophils) from five hypereosinophilic donors activated with 5.0 microM ionophore A23187 for 15 min at 37 degrees C in the presence of 50 mM L-serine released 69 +/- 28 and 1.5 +/- 0.8 (mean +/- SEM) ng of LTC4 and LTB4, respectively, per 10(6) cells; ratios of LTC4 to LTB4 ranged from 16 to 149. Eosinophils stimulated with ionophore (2.5 microM) or phorbol myristate acetate (1 microgram per ml) metabolized exogenously added LTC4 to products that coeluted on reverse-phase high-performance liquid chromatography with synthetic S-diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 diastereoisomers, and this metabolic inactivation was inhibited by L-serine or catalase. Ionophore-activated eosinophils purified from three normal donors also preferentially generated LTC4 (38 +/- 3 ng per 10(6) cells) relative to LTB4 (6.0 +/- 3.1 ng per 10(6) cells), whereas neutrophils from the same donors released LTB4 (48 +/- 21 ng per 10(6) cells) in a greater than 7-fold excess to LTC4. The predominant production by human eosinophils of LTC4 with its potent smooth muscle spasmogenic and vasoactive properties may contribute to the pathobiology of allergic and other diseases associated with eosinophilia.