Cholesterol 24‐hydroxylase is a cytochrome P450 (CYP46A1) that is selectively expressed in the brain and is responsible for the majority of cholesterol turnover in the central nervous system. Mice deficient in 24‐hydroxylase exhibit impaired learning and defective hippocampal long‐term potentiation, suggesting that the metabolism of cholesterol by this enzyme is required for learning and memory formation. To determine where in the neuron cholesterol turnover was taking place, monoclonal antibodies directed against 24‐hydroxylase were generated by immunization of mice with recombinant protein and used to detect the enzyme in brain homogenates, cultured neurons, and histological sections. 24‐Hydroxylase was localized to the endoplasmic reticulum and was distributed throughout the cell bodies and dendrites of multiple types of neurons; the enzyme was not detected in axon terminals or in the cells of 24‐hydroxylase knockout mice. 24‐Hydroxylase was highly expressed in pyramidal neurons of the hippocampus and cortex, in Purkinje cells of the cerebellum, and in hippocampal and cerebellar interneurons. Within the retina, 24‐hydroxylase was detected in ganglion cells and some but not all cells of the inner nuclear layer. These findings reveal the microsomal localization of 24‐hydroxylase and provide subcellular insight into cholesterol turnover in the brain. J. Comp. Neurol. 507:1676–1693, 2008. © 2008 Wiley‐Liss, Inc.