Schwanns cell (SC) was isolated from sciatic nerve of adult rat with Wallerine degeneration. After culture, SC-serum free culture media (SCSFCM) was obtained. By ultrafiltration with PM-10 Amicon Membrane, electrophoresis with DiscPAGE,and electrical wash-out with Biotrap apparatus, D-band protein was isolated from the SC-SFCM. The D-band protein in the concentration of 25ng/ml could affect the survival of the spinal anterior horn neuron in vitro, prominently and itsactivity was not changed after being frozen. The molecular weight of the protein ranged from 43 to 67 Kd. The D-band protein might be a neurotrophic substancedifferent from the known SCderived neurotrophic factors (NTF). Its concentration with biological activity was high enough to be detected. The advantages of MTT in assessment of NTF activity were also discussed.