Background: Suramin is a polysulfonated urea recently tested in clinical trials as an anticancer agent. Purpose: To define tumor types for further clinical testing of suramin, we assessed the in vitro activity of suramin against fresh human tumor specimens. Methods: Inhibition of tumor colony formation (human tumor clonogenic assay [HTCA] method) and inhibition of tritiated thymidine incorporation (TTI method) were used as indicators of drug sensitivity. Results: With the use of the HTCA method, 80% or more of carcinomas of the colon, endometrium, kidney, lung (non-small-cell), and ovary as well as malignant melanoma and mesothelioma were sensitive to 200/mL of suramin by continuous exposure. Suramin‘s antitumor activity was dose dependent, and it was less effective when tested at concentrations of 50μg/mL or less. With the TTI method, the more slowly growing tumors (breast cancer, colon cancer, multiple myeloma, non-Hodgkin’s lymphoma, prostate cancer, and sarcoma) appeared to be less sensitive to suramin. However, when the two assay methods were directly compared in melanoma and ovarian cancer specimens, individual tumors were generally more sensitive to suramin (greater inhibition relative to control) using the HTCA method, whereas the TTI method appeared to underestimate suramin‘s antitumor activity. There was no significant difference in the activity of suramin when tested in the presence of 10% versus 50% serum. Conclusion: These results provide an experimental basis for clinical evaluations of suramin therapy in patients with colon, endometrial, kidney, non-small-cell lung, and ovarian cancers as well as malignant melanoma and mesothelioma. (J Natl Cancer Inst 84: 489–494, 1992].