Objective: Hypoxia significantly alters vascular tone in coronary resistance arterioles during prolonged ischemia, potentially through the modulation of endothelial cell metabolism as well as endothelial function. The objective of this study was to test the hypothesis that constitutive nitric oxide synthase (cNOS) is sensitive to oxygen tension and that hypoxia increases the activity of cNOS and nitric oxide production in the porcine coronary microcirculation. Methods: Monocultures of porcine coronary resistance arteriolar endothelial cells (RAEC) were isolated and proven to be endothelium based upon morphology, binding of acetylated LDL, and factor VIII antigen positivity. Cells were exposed to either hypoxia (pO2= 10 mmHg) or normoxia (pO2= 160 mmHg) for varying periods of time. Nitric oxide production was directly measured using a chemiluminescence method, while cNOS enzyme activity was assayed using a fibroblast-report cell method. cNOS protein was quantitated by Western blot analysis using the H32 monoclonal antibody to the endothelial cell constitutive isoform of NOS. Results: Hypoxia significantly augmented A23187-stimulated nitric oxide production [23.77 (1.73) vs 14.94 (0.66) nmol · μg−1 protein, hypoxia vs. normoxia respectively, n = 8, P ` 0.01]. Using the fibroblast reporter cell assay, cNOS activity was increased in RAEC after exposure to hypoxia for 30, 120 and 240 min [normoxia control: 0.16 (0.04) fmol · μg−1 protein; hypoxia: 30 min = 1.00 (0.19), 120 min = 1.08 (0.04), 240 min = 1.26 (0.07) fmol · μg−1 protein (n = 6, p ` 0.01)]. Western blots showed a single band at 135 kDa that was increased in homogenates of cells previously exposed to hypoxia. Conclusions: These experiments demonstrated that the regulation of cNOS is sensitive to oxygen tension. Hypoxia significantly activated constitutive nitric oxide synthase in coronary resistance arteriolar endothelial cells, and this was translated to an increased production of nitric oxide. © 1995 Elsevier Science B.V. All Rights Reserved.