Summary: Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were stud ied with microspectrofluorometry and the pH-sensitive fluorophore 2',7' -bis( carboxyethyl)-5 ,6-carboxyfluore scein. Steady-state pHi was 7.00 ± 0.17 (mean ± SD) and 7.09 ± 0.14 in nominally HC03 --free and HC03 -containing solutions, respectively, and was dependent on extracellular Na + and Cl -. Following an acid transient, induced by an NH] prepulse or an increase in CO2 ten sion, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmollLlmin, in nominally HC03 --free and HC03 --containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na + and was partially inhibited by amiloride or 5-N-methyl-N-iso butylamiloride. In most cells pHi recovery was com-