Abstract Knockout mouse models allow preparation of primary neuronal cultures from distinct brain regions in order to investigate the underlying neuronal pathomechanisms of human metabolic diseases associated with severe, regionally distinct brain pathologies (e.g. Zellweger syndrome, the most severe form of a peroxisomal biogenesis disorder). However, homozygous mouse pups with Zellweger syndrome usually die shortly after birth. Therefore, in this study, we established optimized protocols for the simultaneous preparation and cultivation of serum-free primary neuronal cultures from distinct brain regions (medial neocortex, hippocampus and cerebellum) from individual newborn (P0.5) C57Bl/6J mice. For each of the three types of neuronal cultures, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, mode of trituration, seeding density and composition of the culture medium. As indicated by indirect immunofluorescence using antibodies against NeuN, GFAP and CNPase, the purity of the distinct neuronal cultures was high. The percentage of oligodendrocytes was less than 1% in all neuronal cultures. Only 5% astrocytes were present in cortical, 7% in hippocampal and 10% in cerebellar cultures. Cytosine arabinofuranoside (AraC) treatment reduced the percentage of astrocytes only significantly in hippocampal cultures, however, increased the percentage of apoptotic neurons in hippocampal and cortical cultures.