Carbonic anhydrases (CAs) are ubiquitous enzymes important to many cell types throughout the body. They help determine levels of H(+) and HCO(-)(3) and thereby regulate intracellular and extracellular pH and volume. CA XIV, an extracellular membrane-bound CA, was recently shown to be present in brain and retina. Here, we analyze the subcellular distribution of CA XIV in retina by high-resolution immunogold cytochemistry and show that the distribution in retina (on glial cells but not neurons) is different from that reported for brain (on neurons but not glia). In addition, CA XIV is strongly expressed on retinal pigment epithelium (RPE). The specific membrane domains that express CA XIV were endfoot and nonendfoot membranes on Muller cells and astrocytes and apical and basolateral membranes of RPE. Gold particle density was highest on microvilli plasma membranes of RPE, where it was twice that of glial endfoot and Muller microvilli membranes and four times that of other glial membrane domains. Neither neurons nor capillary endothelial cells showed detectable labeling for CA XIV. This enrichment of CA XIV on specific membrane domains of glial cells and RPE suggests specialization for buffering pH and volume in retinal neurons and their surrounding extracellular spaces. We suggest that CA XIV is the target of CA inhibitors that enhance subretinal fluid absorption in macular edema. In addition, CA XIV may facilitate CO(2) removal from neural retina and modulate photoreceptor function.