Cholesterol domains and transport have been well-studied in non-neuronal membranes in contrast to neuronal membranes. The purpose of the experiments reported in this paper was to determine: (1) exchangeable and non-exchangeable cholesterol domains or pools were present in brain synaptosomal membranes; (2) effects of hydrolysis of sphingomyelin on cholesterol pools, that has previously been shown to alter membrane cholesterol in non-neuronal membranes and; (3) sphingomyelin hydrolysis and enzyme activity. Cholesterol pools were determined using cholesterol exchange between radiolabeled small unilamellar vesicles and mouse synaptosomes. Activity of Ca2+ + Mg2+-ATPase and Na+ + K+-ATPase were measured in synaptosomal membranes following treatment with sphingomyelinase. The size of the exchangeable pool of synaptosomal membrane cholesterol was approx 50% of total membrane cholesterol when measured at 37°C. The t 1 2 of cholesterol exchange at 37°C in synaptosomes was approx 10 h. Lowering the incubation temperature to 25°C, significantly reduced the size of the exchangeable pool and significantly increased the t 1 2 of cholesterol exchange. Sphingomyelinase treatment of synaptosomes significantly slowed cholesterol exchange but did not modify the size of the exchangeable pool of cholesterol. Ca2+ + Mg2+-ATPase activity was significantly inhibited by sphingomyelinase treatment as compared to Na+ + K+-ATPase activity. Cholesterol domains were described in neuronal tissue and the size and kinetics of those pools were altered by temperature-induced changes in fluidity and hydrolysis of sphingomyelin. Sphingomyelinase-induced changes in Ca2+ + Mg2+-ATPase activity were not affected by hydrolysis of sphingomyelin but appeared to be associated with a reduction in cytofacial phosphatidylinositol.