Neurotrophins signal via Trk tyrosine kinase receptors. NGF is the cognate ligand for TrkA, BDNF for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because NT-3 interactions with TrkA are biologically relevant, we aimed to define the TrkA “hot spot” functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine rich subdomains (D1 and D3), flanking a leucine rich subdomain (D2), and two immunoglobulin-like subdomains IgC1 (D4) and IgC2 (D5). Previously, the D5 subdomain was defined as the primary ligand binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TrkA-D5 hot spots). Here, binding studies with truncated and chimeric extracellular subdomains show that TrkA-D5 also comprises an NT-3 docking and activation “hot spot” (site 1), and competition studies show that the NGF and NT-3 ”hot spots“ on TrkA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3 binding/allosteric site on TrkA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic, but non-competitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and ”hot spots”.