In order to define the molecular anatomy of the blood-brain barrier (BBB) that may be relevant to either barrier or transport function, proteins that are overexpressed in the cerebral microvessels should be identified. We used differential display to identify novel proteins that are overexpressed or unique to the BBB. DNA sequence analysis is one of the differentially expressed transcripts showed that it is highly homologous with the ATPase class I, type 8B, and member 1 (ATP8B1) protein and contains an ATPase domain and a phospholipid-binding domain. ATP8B1 is expressed in the BBB microvessels but not brain tissue lacking microvessels. Likewise, ATP8B1 was enriched in BBB microvessels similar to glucose transporter 1. Immunohistochemistry using an ATP8B1-specific antibody demonstrated preferential staining of the microvessels within the cerebral tissue. These results suggest that ATP8B1, a P-type aminophospholipid translocase, is enriched in cerebral microvessels and may have a role in plasma membrane lipid transport.