A quantitative method consisting of the modification of the zero net flux technique was used to determine the extracellular concentration of ascorbate. A carbon paste electrode (CPE) held at constant potential was combined with a microdialysis probe through which various concentrations of ascorbate were locally infused. In experiments with bilateral CPEs, one of which was combined with a dialysis probe, no difference was observed in either basal or stimulated ascorbate currents. Addition of different concentrations of ascorbate to the perfusion fluid produced rapid changes in the voltammetric current due to ascorbate oxidation. Irrespective of the concentration of ascorbate infused, the current returned to basal level when the perfusion was stopped. The return was found to be more rapid after infusion of concentrations of ascorbate higher than the extracellular concentration than after perfusion with ascorbate-free solutions. This demonstrates that homeostasis of ascorbate occurs in vivo. The calculated point of zero net flux was 416 +/- 66 microM (n = 6). This represents the extracellular concentration of ascorbate.