<p>The sustainable functioning of the purified photosystem I (PSI) pigment-protein complex requires exogenous redox mediators, facilitating the primary electron donor P₇₀₀⁺ reduction and terminal acceptor [F_A/F_B]⁻ oxidation. The redox couple ascorbate/2,6-dichlorophenolindophenol (Asc/DCPIP) was shown to be more efficient, than the couple Asc/<i>N</i>,<i>N</i>,<i>N’</i><i>N’</i>-tetramethyl-<i>p</i>-phenylenediamine (TMPD) both in photosynthetic studies and in biohybrid photovoltaic devices. We investigated the interactions of DCPIP with purified cyanobacterial PSI in the presence of Asc excess under laser flash excitation. Here we show that DCPIP, in contrast to TMPD, competes efficiently as an electron acceptor with the backward electron transfer from [F_A/F_B]⁻ to P₇₀₀⁺ even at micromolar concentrations, indicating accumulation of the oxidized DCPIP under aerobic conditions in the presence of Asc excess. The reduction of P₇₀₀⁺ includes contributions both from reduced DCPIP and semiquinone DCPIP^•−. The rates of P₇₀₀⁺ reduction and [F_A/F_B]⁻ oxidation by DCPIP demonstrate complex pH-dependencies, related to changes in protonation state of the mediator and probably to redistribution of electron density between the terminal cofactors F_A and F_B. The rate constants of the electron transfer from Asc, DCPIP and TMPD to P₇₀₀⁺ and of the electron outflow from [F_A/F_B]⁻ to the oxidized forms of these compounds are estimated by kinetic modeling. The obtained data reveal thermodynamic, kinetic and electrostatic factors responsible for the high DCPIP efficiency as electron donor and acceptor for PSI.</p>