The purpose of the present study was to characterize alterations in the adenylyl cyclase (AC), cyclic adenosine 3′,5′-monophosphate (cAMP), and spatial memory function after sustained cerebral ischemia. Sustained cerebral ischemia was induced by injection of 900 microspheres (48 μm in diameter) into the right (ipsilateral) hemisphere of rats. Alterations in the AC and cAMP in the cerebral cortex and hippocampus were examined up to 7 days after the embolism. A decrease in the cAMP content was seen in the ipsilateral hemisphere throughout the experiment. Microsphere embolism (ME) decreased the activity of Ca2+/calmodulin (CaM)-sensitive AC in the ipsilateral hemisphere throughout the experiment, whereas the basal and 5′-guanylyl imidodiphosphate (Gpp(NH)p)-sensitive AC activities were not altered. Immunoblotting analysis of AC subtypes with specific antibodies showed a decrease in the immunoreactivity of AC-I in the ipsilateral hemisphere during these periods. No significant differences in the immunoreactivity of AC-V/VI and AC-VIII were observed after ME. The levels of GTP-binding proteins Gαs, Gαi, and Gβwere unchanged. Furthermore, microsphere-embolized rats showed prolongation of the escape latency in the water maze task determined on the seveth to ninth day after the operation. These results suggest that sustained cerebral ischemia may induce the impairment of the AC, particularly a selective reduction in the AC-I level and activity, coupled with the decrease in cAMP content. This reduction may play an appreciable role in the disturbance in cAMP-mediated signal transduction system, possibly leading to learning and memory dysfunction. © 2002 Wiley-Liss, Inc.