We have identified a cytoplasmic LIM protein, Ajuba, which interacts with the amino terminus of GLT-1, the most abundant plasma membrane glutamate transporter in the brain. Ajuba has a cytoplasmic location when expressed alone in COS cells, but translocates to colocalize with GLT-1 at the plasma membrane when GLT-1 is co-expressed. Ajuba is expressed in cerebellum, cortex, hippocampus, and retina and also in organs outside the CNS. Ajuba is found with GLT-1 in astrocytes, cerebellar Bergmann glia and retinal neurons, and antibodies to Ajuba coimmunoprecipitate GLT-1 from brain. For GLT-1 expressed in COS cells, coexpression of Ajuba did not affect the transporter's Km or Vmax for glutamate. Since Ajuba is known to activate MAP kinase enzymes, and its homologue Zyxin binds to cytoskeletal proteins, we propose that Ajuba is a scaffolding protein allowing GLT-1 to regulate intracellular signaling or interact with the cytoskeleton.