In skeletal muscle, brain-derived neurotrophic factor (BDNF) has long been thought to serve as a retrograde trophic factor for innervating motor neurons throughout their lifespan. However, its localization in mature muscle fibers has remained elusive. Given the postulated roles of BDNF in skeletal muscle, we performed a series of complementary experiments aimed at defining the localization of BDNF and its transcripts in adult muscle. By reverse transcription-PCR, in situ hybridization, and immunofluorescence, we show that BDNF, along with the receptor p75NTR, is not expressed at significant levels within mature myofibers and that it does not accumulate preferentially within subsynaptic regions of neuromuscular junctions. Interestingly, expression of BDNF correlated with that of Pax3, a marker of muscle progenitor cells, in several different adult skeletal muscles. Additionally, BDNF was expressed in Pax7+ satellite cells where it colocalized with p75NTR. In complementary cell culture experiments, we detected high levels of BDNF and p75NTR in myoblasts. During myogenic differentiation, expression of BDNF became drastically reduced. Using small interfering RNA (siRNA) technology to knock down BDNF expression, we demonstrate enhanced myogenic differentiation of myoblasts. This accelerated rate of myogenic differentiation seen in myoblasts expressing BDNF siRNA was normalized by administration of recombinant BDNF. Collectively, these findings show that BDNF plays an important regulatory function during myogenic differentiation. In addition, the expression of BDNF in satellite cells is coherent with the notion that BDNF serves a key role in maintaining the population of muscle progenitors in adult muscle.