Oxygen exposure for a sufficient duration at high partial pressure results in pulmonary edema in humans and animals. Although the specific mediators of oxygen toxicity are unknown, evidence suggests that oxygen-based radicals such as superoxide anion (O2·) are increased in the lungs in the presence of hyperoxia and contribute to this injury. A series of isomeric prostanoid compounds, the isoprostanes, are formed by the free radical-initiated lipid peroxidation of arachidonic acid (AA). One of these isomers, 8-iso-PGF2α, is elevated in the bronchial alveolar lavage fluid of rats exposed to 90% oxygen for 48 h and is associated with a significant increase in protein accumulation in the pulmonary extravascular space. Alveolar macrophages (AMs) are capable of producing large quantities of (O2·), suggesting a role in pulmonary oxygen toxicity. We hypothesized that isolated rat AMs exposed to hyperoxia generate increased amount of 8-iso-PGF2α. AMs were exposed to air or 90% oxygen for 6, 12, 24, 48, 72, 96, and 120 h in the absence and presence of AA and/or calcium ionophore (A23187) and 8-iso-PGF2α was measured in the culture media. Exposure of primary cultures of AMs to 90% oxygen resulted in a significant increase in 8-iso-PGF2α in the media (25 ± 2 pg/mL) compared with air-exposed controls (14 ± 1 pg/mL). The addition of 10 μM AA and 2 μM A23187 to the culture media resulted in a marked increase in 8-iso PGF2α production by AMs exposed to air and 90% oxygen. However, treatment of AMs with the combination of AA and A23187, followed by exposure to 90% oxygen for 72 h, resulted in a 27-fold increase in 8-iso-PGF2α compared with media alone and 90% oxygen. AMs metabolized free and phospholipid-bound AA to 8-iso-PGF2α' an activity enhanced in the 90% oxygen environment. Finally, acetylsalicylic acid, a cyclooxygenase inhibitor and free radical scavenger, reduced but did not abolish production of 8-iso-PGF2α. This study provides evidence that AMs produce a free radical-mediated isomeric prostaglandin compound that may be involved in pulmonary oxygen toxicity.