Recent evidence indicates that elevated plasma levels of homocysteine are a risk factor for ischemic cerebrovascular diseases. However, little is known about cerebrovascular effects of homocysteine. Homocysteine could impair cerebrovascular function by metal-catalyzed production of activated oxygen species. We studied whether homocysteine, in the presence of Cu²⁺, alters reactivity of cerebral circulation and, if so, whether this effect depends on O₂ generation. In halothane-anesthetized rats the parietal cortex was exposed and superfused with Ringer solution. Cerebrocortical blood flow (CBF) was monitored by a laser-Doppler probe. With Ringer solution superfusion, CBF increased with hypercapnia (+134 ± 7%; PCO₂ = 50-60 mmHg) and topical application of 10 µM ACh (+35 ± 3%), the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 500 µM; +66 ± 6%), or 1 mM papaverine (+100 ± 6%; n = 5). Superfusion with 40 µM Cu²⁺ alone did not perturb resting CBF or responses to hypercapnia, ACh, SNAP, or papaverine (P > 0.05, n = 5). However, superfusion of homocysteine-Cu²⁺ reduced resting CBF (28 ± 4%) and attenuated (P < 0.05) responses to hypercapnia (31 ± 9%), ACh (73 ± 6%), or SNAP (48 ± 4%), but not papaverine. The effect was observed only at 1 mM homocysteine. Cerebrovascular effects of homocysteine-Cu²⁺ were prevented by coadministration of superoxide dismutase (SOD; 1,000 U/ml; n = 5). SOD alone did not affect resting CBF or CBF reactivity (n = 5). The observation that homocysteine-Cu²⁺ attenuates the response to hypercapnia, ACh, and SNAP, but not the NO-independent vasodilator papaverine, suggests that homocysteine-Cu²⁺ selectively impairs NO-related cerebrovascular responses. The fact that SOD prevents such impairment indicates that the effect of homocysteine is O₂ dependent. The data support the conclusion that O₂, generated by the reaction of homocysteine with Cu²⁺, inhibits NO-related cerebrovascular responses by scavenging NO, perhaps through peroxynitrite formation. O₂-mediated scavenging of NO might be one of the mechanisms by which hyperhomocysteinemia predisposes to cerebrovascular diseases.