The ischemic penumbra is a major focus of stroke research. 18F-fluoromisonidazole (18F-FMISO), a positron emission tomography (PET) marker of hypoxic cells, has shown promise as a technique to image the penumbra in humans. Our aim was to delineate the pattern of 18F-FMISO binding in a rat middle cerebral artery transient thread-occlusion model, and correlate this with tissue outcome at 24 hours. We hypothesized that the pattern of 18F-FMISO binding would mimic that seen in humans.Thirty-eight rats underwent 2 hours transient middle cerebral artery (MCA) occlusion, and then received 18F-FMISO at time points from 0.5 to 22 hours post-MCA occlusion and were killed 2 hours later. Autoradiographic assessment of 18F-FMISO binding and assessment (triphenyltetrazolium chloride) of the area of infarction were performed on tissue slices.Until 1 hour after MCA occlusion, 18F-FMISO binding was increased in the entire MCA territory, with little or no infarction visible. Over the next 5 hours, the pattern of binding evolved to a small rim of intensely binding tissue surrounding the infarct core, which itself showed reduced binding compared with the contralateral hemisphere. By 24 hours, there was minimal accumulation of 18F-FMISO binding and a large area of infarction.The pattern of 18F-FMISO binding rats reproduced the pattern seen in humans, consistent with this tracer being a marker of the ischemic penumbra in both species. This technique may have application in studying the ischemic penumbra in animal models, and correlating this with similar studies in humans.