Based upon previous evidence that glutamine formation in astrocytes is pH-sensitive and that raised extracellular K+ alkalinizes astrocytic cytoplasm, it was hypothesized that extracellular K+ might regulate glutamine formation. In this study, the free glutamine content of mouse cerebral astrocytes incubated with 0.1 mM glutamate and 0.1 mM ammonium increased by 80-90% when the extracellular K+ concentration was raised from 3 to 12 mM. The corresponding K(+)-induced intracellular alkalinization of +0.13 pH units only partially reversed a glutamate-induced intracellular acidification of -0.24 pH units. By comparison, adjustment of extracellular pH from 7.4 to 7.8 shifted intracellular pH by +0.25 pH units, fully reversing the glutamate-induced acidification and increasing glutamine content by 120-180%. The effect of K+ on intracellular pH increased to +0.25 pH units in bicarbonate-buffered solution, suggesting that the regulation of glutamine formation by extracellular K+ is enhanced in the presence of bicarbonate.