A hormonally defined medium is described which facilitates the survival of small neurons in primary cultures of mouse cerebellum. The defined medium consists of bovine serum albumin, insulin, transferrin, selenium, thyroxine, and the protease inhibitor aprotinin. About 95% of all cells were identified as neurons using tetanus toxin as a marker in an immunocytochemical assay. They survive for more than 4 weeks, showing a tendency to grow in cell clusters with a dense network of processes. The remaining cells (approximately 5%) were identified as astrocytes by their expression of vimentin and GFA protein and the lack of expression of fibronectin.