The present study was undertaken to investigate the relationship of microglial activation to amyloid beta protein (A beta) deposition, particularly at the early stage. Using single and double immunostaining methods with a panel of microglia markers and antibodies against A beta and amyloid beta protein precursor (APP), we examined the cerebrum and cerebella of both Alzheimer's disease (AD) and non-demented subjects obtained at autopsy. In nondemented, middle-aged subjects that had small amounts of cerebral A beta deposits, approximately 70% of the diffuse plaques contained ramified microglia. However, no evidence of microglial activation was found in diffuse plaques in any of the non-demented subjects. Dual immunostaining of sections of cerebral cortex using antibodies against A beta and major histocompatibility complex class II antigen showed that in AD subjects, approximately 20% of total diffuse plaques contained a few, activated microglia. Most of these plaques were defined as a transitional from between diffuse and primitive plaques. Both primitive and classic plaques in the cerebral cortex of AD subjects consistently contained clusters of activated microglia. Subpial A beta deposits without neuritic changes lacked microglial activation. In the cerebellum, all of the diffuse plaques lacked microglial activation, and activated microglia in the compact plaques were not as hypertrophic as those in cerebral primitive/classic plaques. Our findings indicate that microglial reactions are absent in the early stages of A beta deposition, and it occurs during the transition from diffuse to primitive plaques, when amounts of A beta deposits and the degree of neuritic changes increase.