M easurement of urine to blood (U-B) carbon dioxide tension (P_CO2) gradient during alkalinization of the urine has been suggested to assess distal H⁺ secretion. A fact that has not been considered in previous studies dealing with urinary P_CO2 is that dissolution of HCO₃ in water results in elevation of P_CO2 which is directly proportional to the HCO₃ concentration. To investigate the interrelationship of urinary HCO₃ and urinary acidification, we measured U-B P_CO2 in (a) the presence of enhanced H⁺ secretion and decreased concentrating ability i.e., chronic renal failure (CRF), (b) animals with normal H⁺ secretion and decreased concentrating ability, Brattleboro (BB) rats, and (c) the presence of both impaired H⁺ secretion and concentrating ability (LiCl treatment and after release of unilateral ureteral obstruction). At moderately elevated plasma HCO₃ levels (30-40 meq/liter), normal rats achieved a highly alkaline urine (urine pH > 7.8) and raised urine HCO₃ concentration and U-B P_CO2. At similar plasma HCO₃ levels, BB rats had a much higher fractional water excretion and failed to raise urine pH, urine HCO₃ concentration, and U-B P_CO2 normally. At a very high plasma HCO₃ (>50 meq/liter), BB rats raised urine pH, urine HCO₃ concentration, and U-B P_CO2 to the same levels seen in normals. CRF rats failed to raise urine pH, urine HCO₃, and U-B P_CO2 normally at moderately elevated plasma HCO₃ levels; at very high plasma HCO₃ levels, CRF rats achieved a highly alkaline urine but failed to raise U-B P_CO2. Dogs and patients with CRF were also unable to raise urine pH, urine HCO₃ concentration, and U-B P_CO2 normally at moderately elevated plasma HCO₃ levels. In rats, dogs, and man, U-B P_CO2 was directly related to urine HCO₃ concentration and inversely related to fractional water excretion. At moderately elevated plasma HCO₃ levels, animals with a distal acidification defect failed to raise U-B P_CO2; increasing the plasma HCO₃ to very high levels resulted in a significant increase in urine HCO₃ concentration and U-B P_CO2. The observed urinary P_CO2 was very close to the P_CO2 which would be expected by simple dissolution of a comparable amount of HCO₃ in water. These data demonstrate that, in highly alkaline urine, urinary P_CO2 is largely determined by concentration of urinary HCO₃ and cannot be used as solely indicating distal H⁺ secretion.