In vitro systems are frequently used to study mechanisms of mainstream cigarette smoke (MS)-induced lung injury. Traditional methods of exposure involve the capture of MS particulate phase with filter pads or bubbling MS through phosphate buffered saline (PBS) or cell culture medium. Although useful for in vitro experiments, these exposure methods may fail to capture potential interactions between the gas and particulate phases. To better understand the effect of MS on the human airway, in vitro whole smoke exposure systems that utilize freshly generated whole smoke are needed. Here we report the characterization of a new in vitro whole smoke exposure system (Burghart Mimic Smoker-01 (MSB-01)). This system uses a smoke distribution manifold to simultaneously deliver MS to each well of a 96-well plate. Intraday and interday variations for particulate matter deposition were less than 5% and 13% respectively. Cytotoxicity measurements using lung epithelial BEAS-2B cells indicate variations in calculated EC(50) (half maximal effective concentration) values of 13% intraday and 20% interday. Smoke particulate losses and changes in particle size distribution were also analyzed. The data indicate that 45-50% of the MS generated at the smoking ports is lost within the system prior to delivery into the exposure chamber; however, no changes in particle size distribution were detected throughout the system. Overall, the MSB-01 reproducibly delivered mainstream cigarette smoke in a dose dependent manner across the multiwell plate. The MSB-01 is a high throughput system capable of exposing cells to both the MS particulate and gas/vapor phases simultaneously.