Trabectedin (ET-743, Yondelis) is a novel anticancer drug currently undergoing phase II and III investigations. There are various and conflicting reports whether trabectedin is a substrate for P-glycoprotein (P-gp), an important factor in drug disposition and multi-drug resistance (MDR). We have now unambiguously shown that trabectedin is a P-gp substrate by investigating vectorial transport over monolayers of LLC-PK1 pig kidney and Madine-Darby Canine kidney (MDCK) cells and the mdr1a and/or MDR1 transfected subclones. We further characterized the cytotoxic effects and cellular accumulation of trabectedin in these cell lines as well as in a panel of other cell lines with high or moderate expression levels of P-gp. Trabectedin displayed the typical MDR phenotype only in highly P-gp expressing cell lines, but not in cell lines with expression levels more closely conforming to clinical samples, suggesting that P-gp will not confer resistance to trabectedin in cancer patients.