We have systematically optimized the concentrations of 20 components of a previously published serumfree medium (Brewer and Cotman, Brain Res 494: 65‐74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum‐freemedium Supplement, B27, produced neuron survival above 60% independent of plating density above 160 plated cells/mm² For isolated cells (<100 cells/mm²), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm² by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrnous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/ Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron‐specific enolase. Excellent long‐term viability is achieved after 4 weeks in culture with greater than 90%viability for cells plated at 640/mm² and greater than 50 viability for cells plated at 160/mm² Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones. © 1993 Wiley‐Liss, Inc.