The degree of cellular injury within the stroke ischaemic penumbra is controversial. Clinical and experimental studies using the hypoxia tracer fluoromisonidazole (FMISO) have shown retention of this tracer in the penumbra, but cellular outcome has not been well characterised. We hypothesised that macroscopically intact FMISO-retaining penumbral tissues would show evidence of microscopic injury, and that no FMISO retention would be seen in the infarct core. To determine the distribution of FMISO retention, a tritium-labelled tracer (hydrogen-3 FMISO ([(3)H]FMISO)) was administered 5 minutes after induction of 2-hour temporary middle cerebral artery occlusion. Coregistered brain histology and autoradiography at 24 hours revealed marked retention of FMISO within the infarct. However, 48% of the FMISO-retaining tissue was not infarcted. Within this noninfarcted tissue, only 27% (17 of 64) of sampled regions showed no evidence of neuronal loss, whereas 44% (28 of 64) showed injury to >50% of neurons within the sample. To determine whether FMISO retention occurred after the tissue was already committed to infarction, FMISO was administered 4 to 6 hours after the onset of permanent vessel occlusion. Intense FMISO retention was consistently seen throughout the infarct core. In conclusion, FMISO retention occurs both within the ischaemic penumbra and within the early infarct core. Most penumbral tissues show evidence of selective cellular injury.