A technique to guide brain tissue sampling in experimental focal ischemia is described. This technique allows visual identification for accurate metabolic analysis of regions with variable tissue perfusion. After 2.5 hours of unilateral middle cerebral artery occlusion (MCAO) in the rat, intravenous infusion of the vital dye, neutral red (NR), identified three distinct regions: (a) an intensely stained region consisting of the entire contralateral hemisphere and the medial ipsilateral hemisphere; (b) a blanched region in the lateral ipsilateral hemisphere; and (c) a zone of intermediately stained tissue interposed between the medial and lateral ipsilateral regions. Close regional correlation between NR staining and the iodoantipyrine intensity of adjacent brain slices suggests that NR distribution is a qualitative indicator of brain perfusion. Using the NR staining pattern to guide tissue dissection for metabolite analysis of high energy phosphates (adenosine triphosphate and phosphocreatine) and glucose-related metabolites showed that the blanched region contained ischemic tissue, whereas values from the contralateral cortex resembled those of control tissue. The intermediately stained region exhibited a gradient of metabolic perturbation. Tissue near intensely stained regions resembled control tissue, and tissue near blanched regions resembled ischemic tissue. These results confirm the concept of a metabolic penumbra. When present, it occupies only the most medial area of the intermediate region at 2.5 hours after MCAO.