We report the molecular cloning of a new member of the transmembrane-type immunoglobulin superfamily and designate the encoded protein as limitrin, since it localized selectively to glia limitans in mouse brain. Limitrin cDNA was obtained using a subtractive hybridization procedure designed to identify molecules responsible for blood-brain barrier function. Western blots using a limitrin-specific antibody demonstrated that the gene product is expressed significantly in mouse brain and primary murine astrocytes and is distributed in the plasma membrane. Immunohistochemical studies using confocal and electron microscopy clearly demonstrated highly polarized localization in astroglial endfeet in the perivascular region and under the pia mater in vivo. Limitrin is expressed in the spinal cord and in many areas of the brain, but not in the median eminence or subfornical organ (the circumventricular organs), where the blood-brain barrier is lacking. Disruption of the blood-brain barrier by cold injury resulted in a drastic reduction in limitrin expression. Furthermore, during retrieval from cold injury, the increased expression of limitrin in perivascular endfeet correlated with the recovery of angiogenesis in capillaries within the lesion margins. Our results suggest that limitrin is physically and functionally associated with the blood-brain barrier, implying that this protein may be useful as a diagnostic tool of barrier integrity. © 2003 Wiley-Liss, Inc.