The three members of the neurotrophin family (NGF, BDNF and NT-3) are synthesized as large precursor proteins which undergo proteolytic processing to yield biologically active, mature neurotrophic factors. We have used in vitro mutagenesis to examine the pro-region in the NGF precursor protein as a first step towards a general understanding of the role of propeptides in the biosynthesis of neurotrophins. Our results demonstrate that only two small domains within the NGF propeptide are required for the expression and secretion of properly processed and biologically active, recombinant mouse NGF in COS-7 cells. Domain I plays an important role in the expression of active NGF while domain II is involved in proteolytic processing. Both domains are partially conserved between the propeptides of NGF proteins isolated from different species as well as BDNF and NT-3.