Neurotrophins are important for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the Trk family. The extracellular portion of the Trk receptors has been previously proposed to consist of a cysteine-rich motif, a leucine-rich motif, a second cysteine-rich motif followed by two immunoglobulin-like domains. Earlier studies have shown that a major neurotrophin-binding site in the Trk receptors resides in the second immunoglobulin-like domain. Although the individual amino acids in TrkA involved in binding to nerve growth factor (NGF) and those in TrkC involved in binding to neurotrophin-3 have been mapped in this domain, the Trk amino acids that provide specificity remained unclear. In this study, a minimum set of residues in the human TrkC second immunoglobulin-like domain, which does not bind nerve growth factor (NGF), were substituted with those from human TrkA. The resulting Trk variant recruited binding of NGF equivalent to TrkA, maintained neurotrophin-3 binding equivalent to TrkC, and also bound brain-derived neurotrophin, although with lower affinity compared with TrkB. This implies that the amino acids in the second immunoglobulin-like domain that determine Trk specificity are distinct for each Trk.