Heterologous expression systems based on tobacco BY-2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway((R))-compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher (3)H-IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease-deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration-dependent reversal of (3)H-IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.