OBJECTIVE In the present study, we investigate whether diesel exhaust particles (DEPs) cause cytotoxicity and induce inflammation or increase the expression of mucin in immortalized human middle ear epithelial cell lines (HMEECs). Several publications have shown an association between traffic-related air pollutants and otitis media. Additionally, DEP have been shown to cause inflammation and an allergic response in the airways. METHODS Cell viability following DEP treatment was investigated in HMEECs using the MTT assay. We measured the expression of the inflammatory cytokines TNF-α and COX-2 and the mucin genes MUC5AC and MUC5B using semiquantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS Cell viability tests showed that exposure to more than 80 μg/mL of DEP caused a decrease in cell viability. DEP exposure also increased the expression of MUC5AC, but did not induce the expression of MUC5B in HMEECs. CONCLUSIONS DEP decreased cell viability, induced an inflammatory response, and increased mucin gene expression in HMEECs. These findings support the hypothesis that environmental diesel exposure is a risk factor for otitis media.