Recent in vitro experiments suggest that neurotoxicity of the prion protein is dependent on the presence of microglia. We have studied 11 cases of Creutzfeldt-Jakob disease (CJD) using immunocytochemistry in combination with computerized image analysis to clarify the relationship between spongiform change and microglial activation. MHC class II-positive microglia were almost exclusively confined to cortical gray matter where the neuropil area occupied by these cells exceeded that of controls more than 350-fold. In cortical regions with a bimodal distribution of spongiform degeneration, the presence of class II-positive microglia correlated well with the presence of vacuolation in layer V, but significantly less with spongiform change in layers II and III. In areas where spongiform degeneration affected the entire depth of the cortex, activated microglia were predominantly located in the inner one-half of the cortex or were evenly distributed throughout all cortical laminae. Here, microglia exhibited atypical, tortuous cell processes and occasionally intracytoplasmic vacuoles, suggesting that microglia themselves may become a disease target. Taken together, our results provide indirect evidence against an early causative involvement of microglia in the development of spongiform change. At later stages, however, diseased microglia could produce harmful factors which mediate both astrogliosis and neuronal injury.