Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of pre-degeneration to facilitate enzymatic digestion and recovery of viable cells. To overcome the need for pre-degeneration, we developed a method that allows the isolation of adult rat sciatic nerve SCs immediately after tissue harvesting. This method combines the advantages of implementing a rapid enzymatic dissociation of the nerve fibers and a straightforward separation of cells versus myelin that improves both cell yield and viability. Essentially, the method consists of (1) acute dissociation with collagenase and dispase immediately after removal of the epineurium layer and extensive teasing of the nerve fibers, (2) removal of myelin debris by selective attachment of the cells to a highly adhesive poly-l-lysine/laminin substrate, (3) expansion of the initial SC population in medium containing chemical mitogens, and (4) preparation of cryogenic stocks for transfer or delayed experimentation. This protocol allows for the procurement of homogeneous SC cultures deprived of myelin and fibroblast growth as soon as 3–4 days after nerve tissue dissection. SC cultures can be used as such for experimentation or subjected to consecutive rounds of expansion prior to use, purification, or cryopreservation.