It was previously suggested that the peculiar mechanism of action of the novel anticancer drug Yondelis (ET-743, trabectedin) was due to part of the molecule, units A and B, binding to DNA in the minor groove, causing an alkylation at the N2 of guanine, while unit C protrudes out of DNA, possibly interacting with transcription factors or other DNA binding proteins. To test this hypothesis, we have compared the biological activity and the mode of action of Yondelis with its analogue ET-637, which has the same chemical structure except for the lack of the C ring. Yondelis and ET-637 showed similar cytotoxic potency and cell cycle perturbations. As already reported for Yondelis, the UV-96 cell line, deficient in ERCC-1, was less sensitive to ET-637 than the parental cell line. The binding of Yondelis or ET-637 to DNA-oligonucleotides was demonstrated by gel shift assay and SDS did not reverse the binding. Both compounds blocked the temperature-induced activation of the HSP40 promoter in the range of 1-10 nM. This study indicates that ET-637 acts similarly to Yondelis and demonstrates that the C ring of Yondelis may not be required for its biological activity.