To directly compare biological activities of the neurotrophins NT4 and BDNF in vivo, we replaced the BDNF coding sequence with the NT4 sequence in mice (Bdnf nt4-ki). Mice expressing NT4 in place of BDNF were viable, in contrast with BDNF null mutants, which die shortly after birth. Although the Bdnf nt4-ki/nt4-ki and wild-type Bdnf+/+ alleles yielded similar levels of NT4 and BDNF proteins, NT4 supported more sensory neurons than BDNF and promoted functional synapse formation in cultured hippocampal neurons. Homozygous Bdnf nt4-ki/nt4-ki mice showed reduced body weight, infertility and skin lesions, suggesting unique biological activities of NT4 in vivo. The distinct activities of NT4 and BDNF may result partly from differential activation of the TrkB receptor and its down-stream signals.