Abstract Six ascidian species from the Dutch North Sea coast were screened for cytotoxic activity. Freeze-dried biological material was extracted with solvents of different polarity followed by determination of the cytotoxicity. The most active extracts were further separated using different chromatographic techniques. The microculture tetrazolium (MTT) assay was used to determine the cytotoxicity against two human tumor cell lines: COLO 320 (a colon adenocarcinoma) and GLC 4 (a small cell lung carcinoma). The two cell lines were selected for their different response towards known cytostatics. GLC 4 is sensitive and COLO 320 is resistant to most of the known cytostatics. Three of the species tested yielded interesting fractions. From the in colonial Didemnum lahillei we have isolated a compound which is more active against the COLO 320 cell line (IC 50:33 μg ml −1) than the GLC 4 (IC 50:49 μg ml −1). The structure is currently elucidated. Another colonial species, Aplidium glabrum, yielded a very cytotoxic fraction, with an IC 50 of 5 μg ml −1 against the COLO 320 cell line. From our results it can be concluded that the North Sea could be another interesting source of new compounds with pharmaceutical potential.