Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH4+/NH3. We have studied the entry of NH4+/NH3 into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mm. This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 μm) and by removal of extracellular Cl–. These results strongly suggest that ammonium enters the cell by cotransport of NH4+ with Cl–. Removal of extracellular Na+ did not abolish the NH4+-induced acidification. The NH4+-induced pH change was unaffected when nearly all K+ conductance was blocked with 5 mm Ba2+ showing that NH4+ did not enter through Ba2+-sensitive ion channels. Application of 2 mm NH4+ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH3, we suggest that when NH4+ entered the cells, NH3 left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mm NH4+ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl–. We conclude that NH4+ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH4+(K+)-Cl– cotransporter described in kidney cells.