When sampling inhaled antigens, dendritic cells (DC) must penetrate the tight junction (TJ) barrier while maintaining the TJ seal. In matrix metalloproteinase (MMP)-9–deficient mice, in vivo experiments suggest that migration of DC into air spaces is impaired. To examine the underlying mechanisms, we established a well-defined in vitro model using mouse tracheal epithelial cells and mouse bone marrow DC (BMDC). Transmigration was elicited with either macrophage inflammatory protein (MIP)-1α or MIP-3β in a time-dependent manner. Control MMP-9^+/+ BMDC cultured with granulocyte macrophage–colony-stimulating factor for 7 d showed a 30-fold greater transepithelial migration toward MIP-3β than MIP-1α, indicating a more mature DC phenotype. MMP-9^−/− BMDC as well as MMP-9^+/+ BMDC in the presence of the MMP inhibitor GM6001, although showing a similar preference for MIP-3β, were markedly impaired in their ability to traverse the epithelium. Expression levels of CCR5 and CCR7, however, were similar in both MMP-9^−/− and MMP-9^+/+ BMDC. Expression of the integral TJ proteins, occludin and claudin-1, were examined in BMDC before and after transepithelial migration. Interestingly, occludin but not claudin-1 was degraded following transepithelial migration in both MMP-9^−/− and control BMDC. In addition, there was a > 2-fold increase in claudin-1 expression in MMP-9^−/− as compared with control BMDC. These observations indicate that occludin and claudin-1 are differentially regulated and suggest that the lack of MMP-9 may affect claudin-1 turnover.