Organization and dynamics of neuronal membranes represent crucial determinants for the function of neuronal receptors and signal transduction. Previous work from our laboratory has established hippocampal membranes as a convenient natural source for studying neuronal receptors. In this work, we have monitored the organization and dynamics of hippocampal membranes and their modulation by cholesterol and protein content utilizing location (depth)-specific spin-labeled phospholipids by ESR spectroscopy. The choice of ESR spectroscopy is appropriate due to slow diffusion encountered in crowded environments of neuronal membranes. Analysis of ESR spectra shows that cholesterol increases hippocampal membrane order while membrane proteins increase lipid dynamics resulting in disordered membranes. These results are relevant in understanding the complex organization and dynamics of hippocampal membranes. Our results are significant in the overall context of membrane organization under low cholesterol conditions and could have implications in neuronal diseases characterized by low cholesterol conditions due to defective cholesterol metabolism.