Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase Calpha (PKCalpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKCalpha V3 hinge domain. This motif is also required for a direct association between PKCalpha and beta1 integrin. Efficient binding of beta1 integrin to PKCalpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta1 integrin was shown to block both PKCalpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKCalpha and beta1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.