Lipoxygenase (LO) metabolites of arachidonic acid (AA) regulate vascular tone in mice. Previously, vasoactive LO metabolites such as 11,12,15-trihydroxy-eicosatrienoic acid or 12(S)-hydroxy-eicosatetraenoic acid (12-HETE) were identified in murine arteries. However, the specific LO responsible for the synthesis of these metabolites was not found in vascular endothelium or smooth muscle. The synthesis of 12-LO metabolites depended on the mode of sample preparation. The synthesis of [14C]-AA metabolites was dramatically increased in buffer rinsed, control aortas when compared to heparin-rinsed aortas. We hypothesized that adherent platelets (PLs) are responsible for the synthesis of vasoactive 12-LO metabolites in mouse arteries. Aortic PL adherence and removal of PLs by heparin rinsing were confirmed by immunohistochemical and western immunoblot analysis using specific anti-PL-12-LO and gpIIb/IIIa antibodies. In the presence of indomethacin and L-NA, acetylcholine caused concentration-dependent relaxations in rinsed, control abdominal aortic rings that were preconstricted with thromboxane-mimetic, U46619 (maximum relaxation at 86.4±3.6%) or phenylephrine (maximum relaxation at 81.7±4.1%); however, those relaxations were significantly reduced in heparin-rinsed aortas to 71.6±4.5% (p<0.05) and 48.3±7.2% (p<0.005), respectively. Isolated PLs and rinsed, control aortas showed identical [14C]-AA metabolic patterns. Mass spectrometric identification of AA metabolites produced by rinsed, control aortas or isolated PLs revealed an identical composition consisting of 12-HETE, trioxilins A3 and C3 and hepoxilin A3. Synthetic trioxilin A3, trioxilin C3 and hepoxilin A3 (3 μM) relaxed U46619-preconstricted mesenteric arteries with maximum relaxations of 78.9±3.2; 82.2±5.0 and 20.0±5.1, respectively. These data indicate that adherent PLs contribute to vascular relaxation by producing PL-12-LO-derived relaxing factors. This effect should be taken into consideration when performing functional studies in isolated mouse arteries. Heparin rinsing of isolated arteries is important to remove adherent PLs and eliminate their contributions to vascular tone.