When isolated livers from starved rats are perfused with lactate at constant perfusate pH and PCO2, there is a marked gradient of cell pH (pH1) along the length of the lobular radius, with periportal cells being substantially more alkaline than perivenous cells. In the present studies, the perivenous 21% of the lobular volume was destroyed by retrograde digitonin perfusion, and antegrade perfusion restored, pH1 was determined by 31P-NMR. The remaining periportal cells, the site of gluconeogenesis from lactate, had a substantially higher mean pH1 (7.42) than did the intact liver (7.23). When lactate was removed from the perfusate, mean pH1 decreased to 7.25. The corresponding concentration of cell bicarbonate fell with a half-time of approximately 5 min. When lactate was re-introduced mean pH1 rose to 7.34. We conclude that a major contributor to periportal alkalinity under these conditions is proton consumption during gluconeogenesis from lactate ions.