Aquatic organisms and in particular filter feeders, such as mussels, are continuously exposed to toxicants dissolved in the water and presumably must have adaptations to avoid the detrimental effects from such chemicals. Previous work indicates that activity of ATP binding cassette (ABC) transporters protect mussels against toxicants but the nature of these transporters and the structural basis of protection are not known. Here we meld studies on transporter function, gene expression and localization of transporter protein in mussel gill tissue and show activity and expression of two xenobiotic transporter types in the gills where they provide an effective structural barrier against chemicals. Activity of ABCB/MDR/P-gp and ABCC/MRP type transporters was indicated by sensitivity of efflux of the test substrate calcein-AM to both the ABCB inhibitor PSC833 and ABCC inhibitor MK571. This activity profile is supported by our cloning of complete sequence of two ABC transporter types from RNA in mussel tissue with a high degree of identity to transporters from the ABCB and ABCC subfamilies. Overall identity rates of the amino acid sequences with corresponding homologues from other organisms were 38 - 50 % (ABCB) and 27 - 44 % (ABCC), respectively. C219 antibody staining specific for ABCB revealed that this transporter was restricted to cells in the gill filaments with direct exposure to the water flow. Taken together, our data demonstrate that ABC transporters form an active, physiological barrier at the tissue-environment interface in mussel gills providing protection against environmental xenotoxicants.