We evaluated the effect of age on eicosanoid production in guinea pig blood. Heparinized blood from 7–10-day, 6-week, or 6-month-old guinea pigs was incubated with 150 μM arachidonic acid (AA) for 5 min, followed by stimulation with A23187 (20 μg/mL) for an additional 10 min at 37°. The reaction was terminated by centrifugation, and the production of plasma leukotriene (LT) B 4 and C 4, prostaglandin E 2 (PGE 2), and thromboxane B 2 (TXB 2) was determined by enzyme-linked immunoassay (ELISA). LTC 4, PGE 2, and TXB 2 formation were unaffected by age. In marked contrast, production of LTB 4 was increased 4- to 5-fold as age increased from 7–10 days (9.51 ± 2.07 ng/mL) or 6 weeks (8.83 ± 1.81 ng/mL) to 6 months (40.57 ± 9.66 ng/mL). To determine the effect of age on the total eicosanoid product profile, blood was stimulated in the presence of [ 14C]AA, and plasma metabolites were separated by reverse-phase high pressure liquid chromatography (RP-HPLC) and quantitated using on-line radiochemical detection. In addition to increased LTB 4 production, a modest increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was also observed in the 6-month-old animals. Previous studies have demonstrated interference of 12-HETE in the immunoassay of LTB 4. Therefore, to validate the authenticity of the plasma leukotriene ELISA measurements, samples were precipitated with methanol and fractionated by RP-HPLC. The fractions co-eluting with [ 3H]LTB 4 or [ 3H]LTC 4 were dried under vacuum and reconstituted in ELISA buffer, and leukotrienes were quantitated. As seen previously, following HPLC purification LTB 4 production remained significantly elevated in the 6-month-old guinea pigs, whereas LTC 4 production was unaffected by age. To further document the selectivity of this effect on LTB 4 production, we evaluated the effect of increasing age on cyclooxygenase or phospholipase A 2 (PLA 2) activity. Neither cyclooxygenase nor PLA 2 activity was elevated as animals matured. In conclusion, the capacity of whole blood to produce LTB 4, but not LTC 4, TXB 2, or PGE 2, was elevated markedly in older animals.