CO2 content can be determined from the Pco2 in an acidified (forces all CO2 into solution) and diluted blood sample. However, Pco2 concentrations measured in conventional blood gas analyzers are only correct for samples with a significant buffer capacity (such as whole blood), so that mixing with the Pco2 in the rinse solution and tubing walls does not significantly change the sample Pco2. This study describes a calibration method and validation data for the Radiometer Medical ABL2 CO2 electrode system to accurately measure unbuffered blood samples used in the determination of blood CO2 content (or other aqueous fluids). Prospective, criterion standard. Laboratory. Blood samples (0.4 mL) were acidified and diluted with 0.2 M lactic acid. After measuring Pco2, CO2 content was calculated using the CO2 solubility coefficient and the dilution factor of 20. CO2 content was determined in a series of sodium carbonate (Na2CO3) solutions spanning the physiologic range of CO2 content. Regression of the measured vs. the actual CO2 content data generated a straight line with a slope of 0.796 and y-intercept of 12.5 (r2 = .99; n = 48). These coefficients were successfully used to correct CO2 content determined in blood samples into which graduated amounts of sodium carbonate were added. This calibration procedure allows accurate measurement of Pco2 in aqueous samples using the Radiometer ABL2 electrode system, and should be applicable to other blood gas analyzers. Necessary syringes and chemicals are readily available, the method is fast and simple, and the sample volume is small. In the practice of critical care medicine, accurate Pco2 measurement in aqueous acidified and diluted blood provides direct determination of blood CO2 content (useful in calculations of modified Fick cardiac output or tissue CO2 production). Determinations of absolute CO2 content in blood requiring complex methodology are not necessary. In addition, accurate measurement of aqueous gastric Pco2 can help determine gastric pH, which is an important marker of tissue perfusion.