Pixel-by-pixel spatiotemporal progression of focal ischemia (permanent occlusion) in rats was investigated using quantitative perfusion and diffusion magnetic resonance imaging every 30 minutes for 3 hours. The normal left-hemisphere apparent diffusion coefficient (ADC) was 0.76 +/- 0.03 x 10(-3) mm(2)/s and CBF was 0.7 +/- 0.3 mL x g(-1) x min(-1) (mean +/- SD, n=5). The ADC and CBF viability thresholds yielding the lesion volumes (LV) at 3 hours that best approximated the 2,3,5-triphenyltetrazolium chloride (TTC) infarct volumes (200 +/- 30 mm(3)) at 24 hours were 0.53 +/- 0.02 x 10(-3) mm(2)/s (30% +/- 2% reduction) and 0.30 +/- 0.09 mL x g(-1) x min(-1) (57% +/- 11% reduction), respectively. Temporal evolution of the ADC- and CBF-defined LV showed a significant "perfusion-diffusion mismatch" up to 2 hours (P < 0.05, n = 11), a potential therapeutic window. Based on the viability thresholds, three pixel clusters were identified on the CBF-ADC scatterplots: (1) a "normal" cluster with normal CBF and ADC, (2) an "ischemic core" cluster with markedly reduced CBF and ADC, and (3) a "mismatch" cluster with reduced CBF but slightly reduced ADC. These clusters were color-coded and mapped onto the image and CBF-ADC spaces. Lesions grew peripheral and medial to the initial ADC abnormality. In contrast to the CBF distribution, the ADC distribution in the ischemic hemisphere was bimodal; the relatively time-invariant bimodal-ADC minima were 0.57 +/- 0.02 x 10(-3) mm(2)/s (corresponding CBF 0.35 +/- 0.04 mL x g(-1) x min(-1)), surprisingly similar to the TTC-derived thresholds. Together, these results illustrate an analysis approach to systemically track the pixel-by-pixel spatiotemporal progression of acute ischemic brain injury.