Objective To separate each protein band from the nerve regeneration conditioned fluid(NRCF）and to study whether there are somenew and unknown neurotrophic factors in the protein bands with a relative molecular mass of 220×103. Methods The silicone nerve regenerationchambers were formed in the sciatic nerve of the 25 New Zealand rabbits (weight,1.8-2.5 kg), and NRCF was taken from it at 1 week after operation. The Nativepolyacrylamide gel electrophoresis (Native-PAGE) was used for separating the proteins from NRCF and detecting the relative molecular mass. The Western blot and ELISA were used to observe whether the protein bands ［220×103 (Band a), (20-40)×103(Band c)］ of NRCF could combine with the antibody of the known antibody of neurotrophic factor (NTF):nerve growth factor(NGF), glial cell-derived neurotrophic factor(GDNF), brainderived neurotrophic factor(BDNF), neurotrophin 3(NT-3)， NT-4, ciliang neurotrophic factor(CNTF). Results Separated by Native-PAGE, NRCF mainly contained two protein bands:Band a had a relative molecular mass about 220×103, and Band c had a relative molecular mass about (20-40)×103. Band a could not combine with the antibodies of the NGF, BDNF, CNTF, and NT-3, but could combine with the antibody of NT-4.Band c could combine with the antibodies of NGF, BDNF, CNTF and NT-3, but could not combine with the antibodies of NT-4 and GDNF. Conclusion The protein bands with a relative molecular mass of 220×103 have ber neurotropic and neurotrophic effects than the protein bands with a relative molecular mass of (20-40)×103, which contains NGF,CNTF, etc. NT-4 just has a weak or no effect on the sympathetic neurone. This indicates that there is a new NTF in the protein bands with a relative molecular mass of 220×103, which only combines with the antibody of NT-4.