To facilitate the development of autologous transplantation techniques with which to test the ability of Schwann cell (ScC) implantations to treat nervous system injury, we have developed a method for procuring large, essentially pure populations of ScCs from adult peripheral nerve. By allowing small explants of peripheral nerve trunk to undergo axonal and myelin breakdown in vitro, rather than dissociating the nerve immediately after harvest, we are able to (1) rid the explant of nearly all fibroblasts and (2) capitalize on the intrinsic ScC mitogenic response to peripheral nerve degeneration. Here, we describe a method that yields up to 98% pure ScC populations from adult rat sciatic nerve (based on cell soma and nuclear morphology, S100 staining, and behavior of dissociated cells on neurites) at cell yields of greater than 2 x 10(4) cells/mg of starting nerve weight. The purification technique was successfully applied to human tissue; human phrenic nerve yielded 98% pure ScC populations at cell yields of 2 x 10(4) cells/mg of initial nerve weight. Similar to neonatally derived ScCs, adult rat cells can be expanded in coculture with dorsal root ganglion (DRG) neurons or in isolation in the presence of glial growth factor and forskolin. Cells expanded indefinitely on DRG neurons, or up to 10 weeks on chemical mitogens, return to quiescence following removal of the mitogenic stimulus. Expanded adult-derived rat ScCs retain functional capacity, as evidenced by their ability to myelinate DRG neurites and to support regeneration of processes from embryonic rat retinal explants.