The aim of the present cross-sectional study was to investigate whether activation of the renin-angiotensin system in renovascular disease affects the cytochrome P450 omega/omega-1 hydroxylase (20-hydroxyeicosatetraenoic acid [20-HETE]) and epoxygenase (epoxyeicosatrienoic acids [EETs]) pathways of arachidonic acid metabolism in vivo, each of which interacts with angiotensin II. Plasma concentration and urinary excretion of 20-HETE and EETs and their metabolites, dihydroxyeicosatrienoic acids, were measured in urine and plasma by mass spectrometry in 10 subjects with renovascular disease, 10 with essential hypertension, and 10 healthy normotensive subjects (control subjects), pair-matched for gender and age. Vascular and renal function were evaluated in all of the subjects. Plasma 20-HETE was highest in subjects with renovascular disease (median: 1.20 ng/mL; range: 0.42 to 1.92 ng/mL) compared with subjects with essential hypertension (median: 0.90 ng/mL; range: 0.40 to 2.17 ng/mL) and control subjects (median: 0.45 ng/mL; range: 0.14 to 1.70 ng/mL; P<0.05). Plasma 20-HETE significantly correlated with plasma renin activity in renovascular disease (r(s)=0.67; n=10; P<0.05). The urinary excretion of 20-HETE was significantly lower in subjects with renovascular disease (median: 12.9 microg/g of creatinine; range: 4.4 to 24.9 microg/g of creatinine) than in control subjects (median: 31.0 microg/g of creatinine; range: 11.9 to 102.8 microg/g of creatinine; P<0.01) and essential hypertensive subjects (median: 35.9 microg/g of creatinine; range: 14.0 to 72.5 microg/g of creatinine; P<0.05). Total plasma EETs were lowest, as was the ratio of plasma EETs to plasma dihydroxyeicosatrienoic acids, an index of epoxide hydrolase activity, in renovascular disease (ratio: 2.4; range: 1.2 to 6.1) compared with essential hypertension (ratio: 3.4; range: 1.5 to 5.6) and control subjects (ratio: 6.8; range: 1.4 to 18.8; P<0.01). In conclusion, circulating levels of 20-HETE are increased and those of EETs are decreased in renovascular disease, whereas the urinary excretion of 20-HETE is reduced. Altered cytochrome P450 arachidonic acid metabolism may contribute to the vascular and tubular abnormalities of renovascular disease.