The peroxisome biogenesis factor, peroxin Pex2p, is an integral membrane protein of peroxisomes. As a step toward elucidating the structure and biological function of Pex2p, we determined the transmembrane topology of Pex2p by expressing epitope-tagged rat Pex2p in COS-7 cells. Pex2p tagged with myc at the C-terminus was detected as a punctate staining pattern, when the cells were permeabilized with 50 mu g/ml of digitonin, under which conditions intraperoxisomal proteins such as PTS1-proteins are inaccessible to exogenous antibodies. N-terminally flag-tagged Pex2p was likewise detected upon the same treatment. These results strongly suggest that both the N- and C-terminal parts of Pex2p are exposed to the cytosol, The transmembrane orientation of Pex2p was also assessed by using rat liver peroxisomes and Pex2p region-specific antibodies, The two types of antibodies used, raised to the N(amino acid residues 1-131) and C-terminal part (residues 226 to the C-terminus), respectively, specifically recognized Pex2p and immunoprecipitated intact, whole peroxisomes. Pex2p was not recognized by the antibodies when the peroxisomes were treated with Proteinase K. Furthermore, in situ crosslinking studies involving bifunctional reagents revealed an apparently dimeric form of Pex2p, Therefore, Pex2p is anchored to the peroxisomal membrane by two membrane-spanning segments, with its N- and C-terminal regions exposed to the cytosol.