In order to define the enzymes responsible for the maturation of the precursors of brain-derived neurotrophic factor (proBDNF) and neurotrophin-3 (proNT3), we have analysed their biosynthesis and intracellular processing by the proprotein convertases furin, PC1, PC2, PACE4, PC5 and its isoform PC5/6-B. In these studies, we utilized a vaccinia virus expression system in either BSC40 or the furin activity-deficient LoVo cells. Results demonstrated that in both cells furin and, to a lesser extent, PACE4 and PC5/6-B effectively process proBDNF and proNT3. Furthermore, we have determined that human proNT3 is sulfated, suggesting that processing of proNT3 occurs following the arrival of the precursor to the Trans Golgi Network.