The cellular prion protein (PrPC) is highly conserved in the evolution of mammals, and therefore, thought to have important cellular functions. Despite decades of intensive research, the physiological function of PrPC remains enigmatic. We carried out a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the transmembrane protein tetraspanin-7 (CD231), as a PrPC interacting protein. We confirmed the interaction between PrPC and tetraspanin-7 by yeast two-hybrid assay, immunofluorescent co-localization, and immunocoprecipitation. Our mutational studies further demonstrated that PrPC specifically binds tetraspanin-7 through the region corresponding to bovine PrP154-182 containing alpha-helix 1. (c) 2007 Elsevier Inc. All rights reserved.