Neurotrophin-6 (NT-6) was identified in the teleost fish Xiphophorus as a new member of the neurotrophin gene family. NT-6 binds specifically the glycosaminoglycan heparin. In this study NT-6 was expressed in a stably transfected mammalian cell line, and in insect cells via a recombinant baculovirus. It was purified to homogeneity and characterized by MS and N-terminal sequencing. NT-6 from both expression systems was proteolytically processed at one of two protease cleavage motifs and was found to be glycosylated. It supported the survival of embryonic chick sensory neurons; half-maximal survival was observed at 100 ng/ml. Furthermore, NT-6 elicited neurite outgrowth in explanted embryonic dorsal root ganglia. Addition of heparin into the medium did not potentiate the activity of NT-6 in survival assays. However, when a sensory ganglion explant was cultured in a collagen gel matrix assay adjacent to a heparin bead coated with NT-6, neurite outgrowth directed towards the bead was observed. This indicated that NT-6 was slowly released from the heparin bead generating a concentration gradient of NT-6 instrumental for axonal guidance in vitro. Thus the interaction of NT-6 with heparin might not be required for the activation of the cellular receptor for NT-6 on responsive cells but rather may serve to control, in vivo, the distribution of NT-6.