This is the first report showing that ����Schwann-spheres���� can be obtained from adult peripheral nerves. Moreover, their differentiation, myelination, and neurite growth promoting properties in vitro suggested their potential use in cell transplantation therapy for the damaged nervous system. DRG neurons were co-cultured with cells derived from intact sciatic 6-B345TTQ nerves or Schwann-spheres using the Bryostatin 1 modified method of Hoshikawa et al. The DRGs were taken from adult mice, dissociated with collagenase and trypsin, and seeded on 8-well chamber slides coated with poly-L-lysine at 200,000 cells per well. Thereafter, 250,000 cells from the spheres or intact nerves were seeded onto the DRG cultures in DMEM/F12 medium. The cocultures were incubated for 2 weeks, and then anti-MBP and anti-bIII-tubulin antibodies were applied, followed by the appropriate secondary antibodies. The majority of the cells in the Schwann-spheres were positive for p75, a marker for immature and non-myelinating Schwann cells, whereas very few cells were positive for P0, a marker for myelinating Schwann cells. We next asked whether the Schwann-spheres could differentiate into mature Schwann cells in vitro. After being cultured for 7 days in differentiation medium, approximately 37% of the total cells had differentiated into P0- positive mature Schwann cells, which had a very similar morphology to the mature Schwann cells derived from adult intact sciatic nerves. Furthermore, to determine the origin of the Schwann-spheres, we induced a contusive sciatic nerve injury in MBP-Cre/Floxed-EGFP mice. In these transgenic mice, transient activation of the MBP promoter induces Cre-mediated recombination, indelibly tagging the MBP-positive mature Schwann cells with EGFP expression. Double immunostaining for GFP and p75 in frozen sections of the distal part of the injured sciatic nerves revealed that most of the GFP-positive cells were positive for p75, whereas very few of the GFP-positive cells in intact sciatic nerves were p75-positive, suggesting that myelinating mature Schwann cells could dedifferentiate to the immature stage after peripheral nerve injury. These EGFP-positive cells could form spheres under floating culture conditions, whereas EGFP-negative cells did not. These findings suggested that the spheres were originally derived from MBP-positive mature Schwann cells in the pre-injury sciatic nerves, and that the spheres contained Nestin-positive immature cells.