In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.