Within the ischemic penumbra, there is a heterogeneous development of cortical intracellular acidosis that is associated with selective neuronal injury. This experiment, which used a rabbit model of moderate focal cerebral ischemia, examined the time course for changes in intracellular brain pH, cortical blood flow, capillary bed density, and mitochondrial function in the ischemic penumbra. After cortical annotation of regions of intracellular acidosis in the ischemic penumbra, the animals underwent transcardiac carbon black perfusion for measurement of capillary bed density. Analysis of variance and Pearson's correlation coefficients were used to determine the relationship between capillary bed density, brain intracellular pH, mitochondrial function, and cortical blood flow. Thirty minutes after the onset of ischemia, cortical blood flow declined from 46+/-2 to 22+/-1 mL/100gm/min (P<.01) in all groups. The overall cortical intracellular brain pH measured 6.78+/-.01 compared with a preischemic value of 6.98+/-.01 (P<.05). Within this moderately ischemic cortex, there were small regions (1,000 to 45,000 mum(2)) of increased acidosis, meauring 6.68+/-.01, not associated with focal changes in cortical blood flow, occurring within 15 minutes of ischemia and persisting throughout the ischemic period. Capillary bed density progressively declined with ongoing ischemia occurring after the development of acidosis. For example, capillary bed density in preischemic controls was 338+/-6/mm(2), whereas after 1 hour of ischemia, it measured 147+/-12/mm(2), at 3 hours 97+/-23/mm(2), and at 6 hours 92+/-16/mm(2). Mitochondrial function was reduced coinciding with the decrease in capillary bed density. These data support the hypothesis that cortical acidosis in the ischemic penumbra facilitates the development of perfusion defects that subsequently lead to mitochondrial dysfunction.