Abstract--Angiotensin II and the arachidonic acid metabolite derived via cytochrome P450 20-hydroxyeicostetraenoic acid promote vasoconstriction and vascular smooth muscle cell (VSMC) proliferation. This study was conducted to determine if 20-hydroxyeicostetraenoic acid contributes to angiotensin II-induced neointimal formation in balloon-injured rat carotid artery. In anesthetized rats, the drugs were infused into the clamped segment of the injured right common carotid artery for 60 minutes. The drug solution and catheter were withdrawn, the common carotid artery was ligated, and blood flow was restored. Exposure of the injured artery to angiotensin II (200 nmol/L) or arachidonic acid (10 µmol/L) increased neointimal thickening at day 14 (intima/media ratio 0.71±0.14 with vehicle versus 1.65±0.10 with angiotensin II or 1.31±0.13 with arachidonic acid; P<0.05). Cytochrome P450 4A1 antisense, but not scrambled, oligodeoxynucleotide (100 nmol/L) reduced angiotensin II-induced or arachidonic acid-induced neointimal thickening (intima/media ratio 0.90±0.07 for angiotensin II and 0.95±0.06 for arachidonic acid). 20-hydroxyeicostetraenoic acid (0.5 µmol/L) also increased neointimal thickening of injured artery (intima/media ratio 1.15±0.03); this was not altered by cytochrome P450 4A1 antisense oligodeoxynucleotide. Angiotensin II, arachidonic acid, and 20-hydroxyeicostetraenoic acid also induced the expression of cytochrome P450 4A and increased the number of CD45-positive cells; the latter effect of angiotensin II and arachidonic acid, but not 20-hydroxyeicostetraenoic acid, was diminished by cytochrome P450 4A1 antisense oligodeoxynucleotide. These data suggest that arachidonic acid metabolites derived via cytochrome P450 4A, most likely 20-hydroxyeicostetraenoic acid, mediate angiotensin II-induced neointimal thickening in injured rat carotid artery.