<p>ATP-binding cassette (ABC) drug transporter proteins are expressed at the level of the human pulmonary epithelium. This protein superfamily is known to clinically affect the pharmacokinetics of their substrates in tissues such as the intestine or liver. In contrast, it is not yet entirely understood to what degree ABC transporters contribute as drivers of pulmonary distribution. In recent decades, a number of in vitro studies have been conducted to elucidate the role of ABC transporter in the human lung including human derived cell lines, primary cells and human lung tissue. Results indicate the functional expression of ABC transporters in vitro. However, ABC expression patterns vary across the different cells mimicking upper and lower airways. Since the lung is a target for a variety of drug classes, it is of importance to understand the influence of ABC transporters to pulmonary drug disposition and local drug exposure levels. This review gives an overview over conducted in vitro studies that do not focus only on ABC transporters’ expression but also on assessing their functional activity. Moreover, we pinpoint important aspects that need to be taken into account when conducting these in vitro bidirectional transporter efflux studies. This contributes to a better understanding of the importance of these pulmonary in vitro studies and provides guidance on how to interpret and conduct these studies.</p>