Insulin is the cornerstone of therapy for diabetic ketoacidosis because it causes the rate of ketoacid production to fall; this action takes several hours to occur. Insulin also causes H+ to be transported from the intracellular fluid to the extracellular fluid in vitro. The purpose of this study was to determine if insulin led to the acute export of H+ from the intracellular fluid in vivo. If so, we wished to determine if this also occurred during chronic metabolic acidosis, to quantitate the magnitude of the H+ shift, and to evaluate the mechanisms involved. The administration of low- or high-dose insulin to normal dogs and high-dose insulin to dogs with chronic metabolic acidosis caused the concentration of bicarbonate in plasma to decline by close to 3 mmol/l. The PCO2 fell by close to 15% in all three groups of dogs, so one component of the fall was due to hyperventilation. As the pH of blood did not change, a primary metabolic acidosis also occurred. The fall in bicarbonataemia was not due to net accumulation of organic acids or to a loss of bicarbonate or organic anions in the urine. Taken together, insulin, when given at doses used to treat diabetic ketoacidosis, might induce a significantly greater degree of acidaemia in the extracellular fluid acutely after it is given.