We describe a simple, rapid and highly selective protocol for the primary culture of Schwann cells in vitro from freshly dissociated adult rat nerve. The protocol is based on a selective culture medium comprising both mitogens (forskolin and optionally N2 supplement plus bovine pituitary extract), to stimulate growth of Schwann cells, plus an inhibitory substrate to simultaneously restrict fibroblast overgrowth (D-valine), contained in DMEM. This protocol differs from other available methods in that it uses the preferential capacity of Schwann cells to metabolize D-valine because of the difference in expression of a D-amino acid oxidase (DAAO) enzyme between Schwann cells and fibroblasts plus the presence of a selective mitogen to stimulate growth of Schwann cells. This permits derivation of highly pure Schwann cells directly from fresh adult nerve. Average Schwann cell purities of 97% can be achieved after 19 d without pre-degeneration, purification or antimitotic steps.