Values for pK1' were determined from pH measured at 37 degrees C with three blood-gas analyzers and from calculated pco2 values in 443 freshly separated plasmas, tonometered at 37 degrees C. Plasma was taken from healthy volunteers, seriously ill patients, and hyponatremic patients. pK1' values varied by considerably more than 0.06 in healthy volunteers as well as in very ill patients, and bicarbonate concentrations ([HCO3]p) calculated by blood-gas analyzers based on the pK1' value of 6.1 could be in error by some +/- 60%. pK1' was similarly determined for tonometered (37 degrees C) replicate dilutions of plasma samples. By adding weighed amounts of dry NaCl and NaHCO3 to the diluted samples we increased the Na+ concentration to approximately 150 mmol/L and bicarbonate concentrations to values ranging from approximately 2.5 to approximately 52.5 mmol/L. pK1' values decreased when [HCO3]p was increased in dilutions of plasma kept at constant ionic strength. At any given [HCO3]p, pK1' values were higher at high than at low values of pco2.