CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin α3β1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin α3β1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin α3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin α3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin α3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin α3β1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin α3β1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly¹⁷⁶/Gly¹⁷⁷, Leu¹⁹¹ and Gln¹⁹⁴ comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin α3β1 demonstrated that the segment from Cys¹⁸⁵ to Cys¹⁹², including Leu¹⁹¹, was involved in the stable association of CD151 with integrin α3β1, as was the Gln¹⁹⁴-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu¹⁹¹ and Gln¹⁹⁴, comprise an interface between CD151 and integrin α3β1, and, along with the epitope including Gly¹⁷⁶/Gly¹⁷⁷, are concealed by bound integrin.