Due to the fact that almost all pharmacologic reagents (probe substrates and inhibitors) for drug transporters are not sufficiently selective, there is an unmet need for more definitive in vitro test systems for drug transporters. To address this need, lentiviral vectors expressing shRNA targeting individual human efflux transporters were created. Human Caco-2 cells, derived from a colon adenocarcinoma, were exposed to the vectors, and clones were selected with substantial and stable knockdown of the apical efflux transporters P-gp (MDR1, ABCB1), BCRP (ABCG2), or MRP2 (ABCB2). Various applications of the knockdown cell lines (CPT-P1, P-gp knockdown; CPT-B1, BCRP knockdown; CPT-M1, MRP2 knockdown) have been characterized. Substrates of P-gp and BCRP are identified by performing bidirectional monolayer transport assays with test compound in parental and knockdown cells and comparing efflux ratios in one line vs. the other. The transporters involved in the biliary efflux of a test compound is/are identified in a similar manner, but with the entire panel of knockdown cell lines. Inhibition of BCRP is evaluated in the P-gp knockdown cells using cladribine, a relatively specific BCRP probe substrate whose off-target interaction with P-gp (detected with MDR1-MDCK cells) could confound the interpretation of results obtained in parental Caco-2 cells. False positive and false negative results obtained with other test systems are avoided through the use of the knockdown cells. Finally, the use of knockdown cells with a complement of relevant human uptake transporters enables the elucidation of complex interactions among uptake and efflux transporters, which has been studied with the knockdown cells. These applications will be covered in this presentation.