AbstractA number of studies have provided physiological evidence for extracellular carbonic anhydrase (CA) in brain. Association of extracellular CA with glia has been limited to functional studies of gliotic slices and retinal Muller cells. While astrocytes contain intracellular CA, there has been no direct evidence for surface CA on these cells. In fact, some morphological studies suggest that the extracellular CA in brain parenchyma resides on neurons, not glia. There has been no functional demonstration of extracellular CA activity on CNS neurons, however. Here we capitalized on the H+ dependence of inward lactate transport to reveal functional extracellular CA activity on cultured astrocytes and acutely isolated hippocampal pyramidal neurons. Exposure to 20 mM L‐lactate produced a rapid acidification of astrocytes that was reversibly blocked by 10 μM benzolamide. The lactate‐induced acidification (LIA) was also blocked by a dextran‐conjugated CA inhibitor. In CO2/HCO3−‐free, HEPES‐buffered media, the LIA was largely unaffected. Acutely dissociated hippocampal pyramidal neurons underwent a similar LIA that was reversibly blocked by benzolamide. Surface CA is likely to facilitate lactate transport by enabling rapid replenishment (i.e., buffering) of surface H+ required for inward lactate‐H+ cotransport. These results demonstrate functional surface CA for the first time on individual mammalian astrocytes and neurons and suggest that this enzyme may play a role in the utilization of monocarboxylate substrates such as lactate and pyruvate by the brain. GLIA 41:415–419, 2003. © 2003 Wiley‐Liss, Inc.