The binding of [3H]serotonin to mouse brain crude membrane and synaptosomal membrane preparations was investigated as a function of membrane fluidity changes by lipids. The microviscosity (eta) of the synaptic membranes was increased by in vitro incubation with either cholesteryl hemisuccinate or stearic acid, resulting in an up to 5-fold increase in the specific binding of [3H]serotonin. Serotonin binding increased progressively until it reached a maximum at 1.75 relative eta units; then it declined. Fluidization of membrane lipids, by treatment with lecithin or linoleic acid, caused a small but significant decrease in serotonin binding. These observations are compatible with the concept of vertical displacement of membrane proteins, indicating that in the untreated brain tissue the accessibility (Bmax) of serotonin receptor binding sites constitutes only a fraction (about 20%) of the potential binding capacity stored in the membrane. Scatchard plots of [3H]serotonin binding, at different eta values, indicate a continuous change in the binding affinity (Kd) of serotonin to its receptor, concomitant with changes in its accessibility. These results may have important implications for physiological processes in the central nervous system, which are associated with modulation of membrane lipids, such as aging. In addition, the regional heterogeneity and plasticity of receptors may be accounted for by differences in membrane lipid fluidity. It was found here that various brain regions differ markedly in their membrane lipid viscosity.