Ascorbate is the major electron donor to a transplasma membrane electron transport (tPMET) system that was originally identified in human erythrocytes. This plasma membrane redox system appears to transfer electrons from intracellular ascorbate to extracellular oxidants (e.g., non-transferrin-bound iron). Though this phenomenon has been observed in nucleated cells, its mechanism and regulation are not well understood. Here we have examined both facets of this phenomenon in K562 cells and primary astrocyte cultures. Using ferricyanide as the analytical oxidant we demonstrate that tPMET is enhanced by dehydroascorbate uptake via facilitative glucose-transporters, and subsequent accumulation of intracellular ascorbate. Additionally, we demonstrate that this stimulation is not due to ascorbate that is released from the cells, but is dependent only on a restricted intracellular pool of the vitamin. Substrate-saturation kinetics suggest an enzyme-catalysed reaction across the plasma membrane by an as-yet-unidentified reductase that relies on extensive recycling of intracellular ascorbate. Inhibition of ascorbate-stimulated tPMET by the Na+/H+-exchange inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride, which is diminished by bicarbonate, suggests that tPMET activity may be regulated by intracellular pH. In support of this hypothesis, tPMET in astrocytes was significantly inhibited by ammonium chloride-pulse-induced intracellular acidification, while it was significantly stimulated by bicarbonate-induced intracellular alkalinisation. These results suggest that ascorbate-dependent tPMET is enzyme-catalysed and is modulated by NHE activity and intracellular pH.