To evaluate the utility of the urinary-minus-blood partial pressure of carbon dioxide (U-B PCO2) gradient for the diagnosis of distal renal tubular acidosis in neonates, we measured the U-B PCO2 gradient corresponding to different urinary bicarbonate concentrations in 40 neonates. The U-B PCO2 gradient in these neonates had a significant linear relationship to the urinary bicarbonate concentration. When the urinary bicarbonate concentration was > 10 mmol/L, in all the neonates the U-B PCO2 could be increased above the 20 mm Hg level. We conclude that it is appropriate to determine the U-B PCO2 gradient as an index of distal urinary acidification and that it is a necessary test for diagnosis of distal renal tubular acidosis in neonates.