Previous studies evaluating the mechanism of renal HCO-3 reabsorption have assumed equilibrium between systemic arterial blood and tubular fluid PCO2. We have recently reported that the PCO2 in proximal and distal tubular fluid as well as the stellate vessel significantly exceeded arterial PCO2 by 25.9 +/- 0.92 mm Hg. The purpose of this study was to determine directly, for the first time, pH, PCO1, and total CO2 concentration in the accessible structures of the rat renal cortex with both microelectrodes and microcalorimetry. In addition, the concentrations of chloride and total CO2 were compared in the stellate vessel. The data demonstrate that: (a) values for total [CO2] in both the proximal tubule and stellate vessel calculated from in situ determination of pH and PCO2 closely agree with the measured values for total [CO2]: (b) values for chloride concentration in the stellate vessel are significantly less than the corresponding values in systemic plasma (delta[Cl-] = 5.6 meq/liter); and (c) the rise in [HCO-3] from systemic to stellate vessel plasma closely approximates the observed reciprocal fall in [Cl-] in this structure.