1. The content of adenosine triphosphate (ATP) and basal and bradykinin-stimulated production of prostacyclin and endothelium-derived relaxing factor (EDRF) was measured in primary cultures of porcine aortic endothelial cells under normoxic (14.4% O2) and hypoxic (2.8% O2) conditions, and following treatment with rotenone and 2-deoxy glucose, which inhibit oxidative and glycolytic metabolism, respectively. 2. ATP content and basal and bradykinin-stimulated production of prostacyclin were similar under normoxic and hypoxic conditions. EDRF production, assessed as endothelial guanosine 3':5'-cyclic monophosphate (cyclic GMP) content, was also similar under both conditions. 3. Treatment with rotenone (0.3 microM) had no effect on ATP content or basal or bradykinin-stimulated production of prostacyclin or of EDRF, measured as endothelial cyclic GMP content. Elevation of cyclic GMP content by atriopeptin II was also unaffected. 4. Treatment with 2-deoxy glucose (20 mM) in glucose-free Krebs solution lowered ATP content, reduced bradykinin-stimulated production of prostacyclin and abolished the bradykinin-stimulated elevation of cyclic GMP content. Resting production of prostacyclin was unaffected but basal content of cyclic GMP was lowered in some experiments. Elevation of cyclic GMP content by atriopeptin II was abolished. 5. Combined treatment with rotenone (0.3 microM) and 2-deoxy glucose (20 mM) lowered ATP content more than with 2-deoxy glucose alone. Basal production of prostacyclin rose slightly and bradykinin-stimulated production was powerfully inhibited. Basal content of cyclic GMP was unaffected, but bradykinin-stimulated production was abolished. Elevation of cyclic GMP by atriopeptin II was also abolished. 6. Cascade bioassay experiments using endothelium-denuded rings of rabbit aorta as a detector system confirmed that bradykinin-stimulated production of EDRF was blocked by 2-deoxy glucose, but not by rotenone. 7. These data indicate that porcine aortic endothelial cells in culture operate under mainly glycolytic metabolism and this probably explains why production of prostacyclin and EDRF is unaffected under hypoxic conditions. They also indicate that glycolytic metabolism is required for agonist-stimulated production of prostacyclin and EDRF by these cells.