AbstractCancer metastasis is a multiple‐step process that involves the regulated interaction of diverse cellular proteins. We recently reported that the expression of tumor‐associated antigen L6 (TAL6) promoted the invasiveness of lung cancer cells and was inversely correlated with disease‐free survival of squamous lung carcinoma patients. We now report that CD13 (aminopeptidase N) can associate with TAL6 and can enhance cancer cell migration. CD13 was shown by coimmunoprecipitation to associate in vitro with TAL6 on several cancer cell lines and to associate in vivo by antibody‐mediated copatching immunofluorescence. CD13 was selectively expressed on highly invasive CL1‐5 lung cancer cells as compared to poorly invasive CL1‐0 lung cancer cells. The role of CD13 aminopeptidase activity in regulating cell motility was investigated with chemical inhibitors, specific antibodies and a catalytically inactive CD13 protein. Inhibition of CD13 aminopeptidase activity by nontoxic concentrations of leuhistin modestly decreased the migration of CL1‐5 cells. In contrast, binding of CD13 by specific antibodies significantly reduced both the migration and the invasion of CL1‐5 cells. Poorly invasive CL1‐0 cells that stably expressed CD13 displayed significantly (p ≤ 0.0005) enhanced cell migration (300% of control). Expression of an enzymatically inactive CD13 mutant on CL1‐0 cells also significantly (p ≤ 0.0005) enhanced cell migration (200% of control). Our results show that TAL6 and CD13 can form a complex on lung cancer cells, that these molecules can modulate cell migration and invasion and that the influence of CD13 on cell motility did not strictly depend on its aminopeptidase activity. © 2005 Wiley‐Liss, Inc.