Endocrine disrupting chemicals (EDCs) are considered to cause testicular toxicity primarily via interference with steroid hormone function. Alternatively, EDCs could possibly exert their effects by interaction with ATP-binding cassette (ABC) transporters that are expressed in the blood-testis barrier. Here, we investigated the effects of bisphenol A (BPA), tetrabromobisphenol A (TBBPA), bis(2-ethylhexyl) phthalate (DEHP), mono(2-ethylhexyl) phthalate (MEHP), perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) on breast cancer resistance protein (BCRP), multidrug resistance protein 1 and 4 (MRP1,4), and P-glycoprotein (P-gp) using membrane vesicles overexpressing these transporters. BPA solely inhibited BCRP activity whereas TBBPA, PFOA and PFOS inhibited all transporters tested. No effect was observed for the phthalates. Using transporter-overexpressing Madin-Darby canine kidney (MDCKII) cells, we show that BPA and PFOA, but not TBBPA, are transported by BCRP, whereas none of the compounds were transported by P-gp. To investigate the toxicological implications of these findings, testosterone secretion and expression of steroidogenic genes were determined in murine Leydig (MA-10) cells upon exposure to the selected EDCs. Only BPA and TBBPA concentration-dependently increased testosterone secretion by MA-10 cells to 6- and 46-fold of control levels, respectively. Inhibition of the Mrp's by MK-571 completely blocked testosterone secretion elicited by TBBPA, which could not be explained by coinciding changes in expression of steroidogenic genes. Therefore, we hypothesize that transporter-mediated efflux of testosterone precursors out of MA-10 cells is inhibited by TBBPA resulting in higher availability for testosterone production. Our data show the toxicological and clinical relevance of ABC transporters in EDC risk assessment related to testicular toxicity.