The role of neurotrophins in thalamic axon growth was studied by culturing embryonic rat thalamus on collagen-coated substrate or fixed cortical slices in the presence of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Both BDNF and NT-3 promoted axonal growth, but the axonal growth-promoting activity depended on culture substrates. Axonal growth on collagen-coated membrane was accelerated by BDNF, but not by NT-3. In contrast, axonal outgrowth on fixed cortex was significantly enhanced by NT-3, but not by BDNF. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cultured thalamic cells demonstrated that culture substrates did not alter the expression of their receptors, trkB and trkC. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining further demonstrated that axonal growth promoted by neurotrophins was not due to reduction of cell death. Measurement of the developmental changes in BDNF and NT-3 levels revealed that, in contrast to the rapid elevation of BDNF after the arrival of thalamocortical axons to their target layer, the regulation of NT-3 protein accompanies the phase of their outgrowth in neocortex. These findings suggest that BDNF and NT-3 promote thalamic axon growth in different manners in terms of substrate dependency and developmental stage.