The cerebellar Purkinje neuron (PN) serves as an important model in studies of neuronal development in the mammalian central nervous system. Dissociated PN preparations maintained in an in-vitro environment with simplified cellular and biochemical conditions can facilitate molecular analyses of neuronal development. Here we describe a reliable method to prepare dissociated cultures of mouse cerebellar neurons maintained with a serum-free, Dulbecco's modified Eagle's medium/F-12 nutrient-based medium, which facilitates PN survival and dendritic differentiation. The survival of mouse PNs in this culture was maximized when cerebellar cells were (1) taken from prenatal animals, (2) dissociated with papain, and (3) seeded at a density of 5 000000 cells/ml or higher. Dissociated PNs prepared by our method from mice of embryonic day 18 (E 18) reproduced several morphological and electrophysiological changes seen in intact postnatal rodents with similar time-courses. Therefore, our culture method offers a useful model for investigating molecular mechanisms underlying postnatal neuronal development.