Abstract Electrostatic deposition of particles onto the surface of well-differentiated airway cells is a rapid and efficient means to screen for toxicity associated with exposure to fine and ultrafine particulate air pollution. This work describes development and application of an electrostatic aerosol in vitro exposure system (EAVES) with increased throughput and ease-of-use. The modified EAVES accommodates standard tissue culture plates and uses an alternating electric field to deposit a net neutral charge of aerosol onto air-interface cell cultures. Using this higher-throughput design, we were able to examine the time-course (1, 3, 6, 9, and 24 hours post-exposure) of transcript production and cytotoxicity in well-differentiated human bronchial cells exposed to diesel particulate matter at levels of ‘real-world’ significance. Statistically significant responses were observed at exposure levels (∼0.4 μg/cm2) much lower than typically reported in vitro using traditional submerged/resuspended techniques. Levels of HO-1, IL-8, CYP1A1, COX-2, and HSP-70 transcripts increased immediately following diesel particulate exposure and persisted for several hours; cytotoxicity was increased at 24 hours. The modified EAVES provides a platform for higher throughput, more efficient and representative testing of aerosol toxicity in vitro.