Purpose: Patients with pancreatic adenocarcinoma have a poor prognosis due to the extraordinary high invasive capacity of this tumor. Altered integrin and tetraspanin expression is suggested to be an important factor. We recently reported that after protein kinase C activation, colocalization of α6β4 with the tetraspanin CO-029 strongly supports migration of a rat pancreatic adenocarcinoma. The finding led us to explore whether and which integrin-tetraspanin complexes influence the motility of human pancreatic tumors. Experimental Design: Integrin and tetraspanin expression of pancreatic and colorectal adenocarcinoma was evaluated with emphasis on colocalization and the impact of integrin-tetraspanin associations on tumor cell motility. Results: The majority of pancreatic and colorectal tumors expressed the α2, α3, α6, β1, and β4 integrins and the tetraspanins CD9, CD63, CD81, CD151, and CO-029. Expression of α6β4 and CO-029 was restricted to tumor cells, whereas α1, α2, α3, α6, β1, and CD9, CD81, CD151 were also expressed by the surrounding stroma. CD63, CD81, and β1 expression was observed at comparably high levels in healthy pancreatic tissue. α3β1 frequently colocalized and coimmunoprecipitated with CD9, CD81, and CD151, whereas α6β4 colocalized and coimmunoprecipitated mostly with CD151 and CO-029. Notably, protein kinase C activation strengthened only the colocalization of CD151 and CO-029 with p4 and was accompanied by internalization of the integrin-tetraspanin complex, decreased laminin 5 adhesion, and increased cell migration. Conclusion. α6β4 is selectively up-regulated in pancreatic and colorectal cancer. The association of α6β4 with CD151 and CO-029 correlates with increased tumor cell motility.