Introduction: Our current knowledge about the effect of cigarette smoke (CS) in vitro is largely based on exposing cells growing in submersed conditions to CS fractions. Although the particulate and gas–vapor phases (or CS condensate) are still widely used for in vitro studies, they have some important limitations. In particular, the method used for trapping them might alter the chemical composition of each fraction. Some compounds (especially volatile compounds) cannot be trapped quantitatively; the filters or other components of the collection system might leach impurities into the collected material and the solvents used might react with constituents of the smoke fraction. Most importantly, analysis of the individual fractions might underestimate the overall risk attributable to CS through combined exposure to multiple toxicants. In addition, using submersed cell culture systems to test the effect of CS fractions, do not resemble the conditions under which cells are exposed to CS in the human lung. For these reasons, researchers are increasingly using culture systems where cells are exposed to CS at the air-liquid interface (ALI). Only a few exposure systems are presently available that enable CS exposure of living cells at the air–liquid interface, of which one of the most versatile is the Vitrocell® system (Vitrocell® Systems GmbH). To assess its performance and optimize the exposure conditions, we characterized a Vitrocell® 24/48 system connected to a 30-port carousel smoking machine.