Neuronal cell death after brain ischemia may be regulated by activation of cyclin-dependent kinase 5 (Cdk5). In this study, expression of Cdk5 and its activator p35/p25 was examined in human post-mortem stroke tissue and in human cerebral cortical fetal neurons and human brain microvascular endothelial cells exposed to oxygen-glucose deficiency and reperfusion. The majority of patients demonstrated increased expression of Cdk5 and p-Cdk5 in stroke-affected tissue, with about a third showing increased p35 and p25 cleaved fragment as determined by Western blotting. An increase in Cdk5-, p-Cdk5- and p35-positive neurons and microvessels occurred in stroke-affected regions of patients. Staining of neurons became irregular and clumped in the cytoplasm, and nuclear translocation occurred, with colocalization of p35 and Cdk5. Association of Cdk5 with nuclear damage was demonstrated by coexpression of nuclear Cdk5 in TUNEL-positive neurons and microvessels in peri-infarcted regions. In vitro studies showed up-regulation and/or nuclear translocation of Cdk5, p-Cdk5 and p35 in neurons and endothelial cells subjected to oxygen-glucose deficiency, and strong staining was associated with propidium iodide positive nuclei, an indicator of cellular damage. These results provide new evidence for a role of Cdk5 in the events associated with response to ischemic injury in humans.