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Lissoclinolide is a small non-nitrogenous lactone isolated from the marine ascidian Lissoclinum patella. Previous studies of lissoclinolide (isolated from a fungus and an actinomycete) have identified varying activity against both Gram-negative and Gram-positive bacteria. In this study, lissoclinolide was able to inhibit cell growth in various mammalian tumor lines at an average IC50 of 395 nM (determined by MTT conversion after 48-h treatment). Treatment of HCT 116 human colon tumor cells with 2.4 μM lissoclinolide resulted in a strong arrest in the G2/M phase of the cell cycle after 24-h exposure. A daughter cell line lacking p53 showed an identical response while there was a slight increase in cytotoxicity towards a p21 null cell line. Although treatment with 2.4 μM lissoclinolide did not result in apoptosis after 48 h, this arrest was not reversible when drug wash out was attempted. The mechanism of action does not appear to involve tubulin, ubiquitin-specific isopeptidases, p53 or p21. COMPARE analysis in the NCI 60 cell line tumor panel revealed a moderate selectivity towards colon tumor cell lines.