The anticancer activity of ametantrone was investigated in a human tumor cloning assay. Tumor samples were freshly obtained from 105 patients. Cells were exposed for 1 hr to drug concentrations of 1 and 10 μ g/ml. A reduction in the number of tumor colony-forming units by 50% or more was seen in 2 31 breast cancers, 2 25 ovarian cancers, 1 10 primaries of unknown origin, 1 10 melanomas, 2 8 non-small cell lung cancers, 1 5 small cell lung cancers and 1 3 colon cancers. Only three of these in vitro responses were consistently obtained at the probably more relevant concentration of 1 μ g/ml. These findings indicate that low efficacy should be expected in cancer patients with ametantrone. The predictive value of these in vitro phase II data remains to be demonstrated.