Two relatively well characterised kinase signalling pathways are those involving MAPK/ERK and p38/SAPK2, that are known to be activated in vitro by various factors known to increase following stroke, such as glutamate, IL-1 and TNF. The present study was designed to investigate the activation and cellular distribution of phosphorylated-ERK1/2, -p38 and the transcription factor CREB following focal cerebral ischaemia using phosphospecific antibodies. Up to 24 h following transient MCAO (90 min) and 6 h following permanent MCAO, phospho-ERK1/2 staining was markedly increased within the cytoplasm of neuronal perikarya in 'penumbral-like' regions. In contrast, phospho-p38 immunostaining was markedly increased in cells with astrocyte-like morphology in both 'core' and 'penumbral-like' regions. Phospho-p38 staining was also detected in some neurones within 'penumbral-like' regions up to 24 h following transient MCAO. CREB activation was confined to neurones in 'penumbral-like' regions. Increased phospho-p38 immunoreactivity was detected in astrocyte-like cells present in the subcortical white matter ipsilateral to the occluded MCAO, while phospho-CREB and -ERK1/2 staining was localised to cells with the morphological appearance of oligodendrocytes. This study demonstrates phosphorylation, indicative of activation, of both the MAPK and p38 pathways following transient and permanent MCAO. However, each pathway shows a distinct cellular and spatial distribution within ischaemic tissue. Together these data indicate that neuroprotection offered by agents directed towards the ERK1/2 pathway may act directly through protection of neurones and oligodendrocytes, while those directed towards the p38 pathway kinase signalling pathways may be indirectly via inhibition of cytokines and other mediators involved in the brains response to injury.