Although mature neurones do not replicate genomic DNA, some cell cycle-related kinases are aberrantly activated in neurones after ischaemia. As hyper-phosphorylation of retinoblastoma (Rb) protein is the common pathway in mitotic signal cascade, this study investigated the phosphorylation state of the Rb protein as well as its mRNA level in rat brain after transient middle cerebral artery (MCA) occlusion. Immunohisto-chemical analysis revealed that neurones in the sham-operated brain expressed Rb protein without the hyperphosphorylated form. Immunoreactivity for the hyperphosphorylated form of Rb protein progressively increased from 1 h to 3 days after ischaemia in neurones in the MCA territory. Western blot analysis demonstrated a similar change. However, reverse transcription-polymerase chain reaction study revealed that Rb showed no definite change at the mRNA level. These results suggest that Rb protein is progressively hyper-phosphorylated in the brain after ischaemia, which may activate apoptotic mechanisms in neuronal cells of the brain after ischaemia.