We have determined that all four known members of the neurotrophin family, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4), are capable of forming noncovalent heterodimers. The formation of these heterodimers was accomplished by homodimer subunit exchange promoted by treatment with guanidine hydrochloride, urea, low pH, or acetonitrile. In some cases (BDNF and mouse NGF; BDNF and NT-4), generation of the heterodimers was achieved by incubating homodimer mixtures in a neutral buffer at ambient temperature. The formation of heterodimers was in each case detected by nondenaturing gel electrophoresis at pH 7.4. High-performance cation-exchange chromatography was used to separate neurotrophin heterodimers from their parental homodimers. Heterodimers between BDNF and NT-3, BDNF and NT-4, and NT-3 and NT-4 are stable and show only a very small increase in homodimer content after 24 h of incubation at 37 degrees C. In contrast, heterodimers containing NGF subunits undergo gradual rearrangement to the homodimers. Our studies indicate that low pH, acetonitrile, and urea merely increase the neurotrophin subunit exchange rate and decrease the time needed to reach an equilibrium between a heterodimer and its two parental homodimers.