Abstract In the nervous system, alternative pre-mRNA splicing generates the diverse protein machineries needed for cell excitation and synaptic communication. Yet, many questions remain about how these mechanisms are regulated by RNA binding proteins in the environment of differentiated cells and tissues. Here, we describe the preparation and use of splicing active nuclear extracts derived from the cerebellum and cerebral cortex regions of rat brain as a resource for in vitro studies. These tissue-specific extracts promote the neuron-specific pathway of splicing, and display characteristic changes in hnRNP protein function and expression. These extracts can be used in combination with affinity selection and depletion/complementation assays to identify regulatory factors and to characterize their interactions and effects on spliceosome assembly. The ability to prepare extracts from brain regions at a range of postnatal ages provides opportunities to address related questions as a function of cell differentiation. These neuronal extracts may also be valuable for the development of in vitro assays to elucidate other neuron-specific RNA processing pathways, such as 3′ end formation, RNA editing, or miRNA maturation.