During vertebrate development, many neurons depend for survival and differentiation on their target cells. The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF). NGF and the other known members of the neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) are conserved as distinct genes over large evolutionary distances. Here we report the cloning of neurotrophin-6 (NT-6), a new member of this family from the teleost fish Xiphophorus. NT-6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells. The addition of heparin (but not chondroitin) effects the release of NT-6 from cell surface and extracellular matrix molecules. Recombinant purified NT-6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency. NT-6 is expressed in the embryonic valvulla cerebelli; expression persists in some adult tissues. The interaction of NT-6 with heparin-binding molecules may modulate its action in the nervous system.