The acellular nerve segment is thought to be a method of choice for reparation of nerve defect. Repeated periods of freezing and thawing are frequently used for preparation of standard acellular nerve segment in experimental application. It was believed that the basal lamina tubes and endoneurial extracellular matrix molecules survive the procedures and promote the growth of regenerating axons. We used the model of freeze-treated segment of the distal nerve stump to study a molecular content of the endoneurium in absence and presence of Schwann cells. The nerve segment, marked out at the end of distal stump of transected rat sciatic nerve, was repeatedly frozen and thawed (5 times) to evacuate all resident cells. Serial longitudinal frozen sections, 10 microm thick, were incubated with monoclonal antibodies against laminin-1, -2, and -3, fibronectin, thrombospondin, as well as chondroitin sulfate. The position of Schwann cells was detected in subsequent sections or simultaneously by immunofluorescence staining with polyclonal antibody against S-100 protein. The results demonstrated that migrating Schwann cells (S-100+) repopulated the nerve segment from untreated regions of the distal nerve stump. Subsequent sections incubated for the above-mentioned molecules of the extracellular matrix revealed immunostaining only in the regions containing the Schwann cells. Simultaneous (double) immunostaining confirmed that the immunofluorescence for laminins, fibronectin, thrombospondin and chondroitin sulfate is restricted to the presence of Schwann cells. Our results suggest that Schwann cells that are crucial for arrangement of axon-promoting condition synthesize the ECM molecules and support the endoneurium by them without presence of the axons. This supports a critical role of Schwann cell migration for the growth of axons through the acellular nerve segment.