The bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) carries out two distinct reactions, with the dihydrofolate produced by the TS-catalyzed reaction acting as the substrate for the DHFR-catalyzed reaction. Brownian dynamics simulation techniques were used to investigate the possible role of electrostatics in determining efficient channeling of the substrate, by explicitly simulating substrate diffusion between the two active sites. With a substrate charge of -2, almost all (> 95%) substrate molecules leaving the TS active site reached the DHFR active site at zero ionic strength. Under the same conditions, but in the absence of electrostatic effects, successful channeling was reduced to only around 6%: electrostatic effects therefore appear essential to explain the efficient channeling observed experimentally. The importance of substrate charge, the relative contributions of specific basic residues in the protein, the role played by the second monomer of the dimer in channeling and the effects of changing ionic strength were all investigated. Simulations performed for substrate transfer in the opposite direction suggest that channeling in DHFR-TS is not strongly directional and that the role of electrostatics is perhaps more one of restricting diffusion of the substrate than one of actively guiding it from the TS to the DHFR active site. The results demonstrate that electrostatic channeling can be a highly efficient means of transferring charged substrates between active sites in solvent-exposed environments.