The catalytic mechanism of dihydrofolate reductase (DHFR) requires the addition of both a proton and a hydride ion. In Escherichia coli dihydrofolate reductase (ecDHFR) Asp27 is considered as the only group in the active site capable of providing the proton for the reduction of the N5-C6 bond, although it is not clear enough if it takes place directly or through a series of water molecules. In this paper we present a theoretical study of the protonation of N5 of the hydrofolate, the prior step of the hydride transfer in dihydrofolate reductase. Hybrid quantum-mechanical/molecular-mechanicaI (QM/MM) calculations involving a flexible active-site region are used in combination with GRACE software. Two different reaction paths have been found to be feasible. The proposed mechanisms are in agreement with the ordered structure of the X-ray crystallographic water molecules supporting the hypothesis of an indirect proton transfer from Asp27 residue to the N5 atom of the substrate. Asp27 is found to play an important role stabilizing the cationic pteridine ring.