We have investigated the biodistribution, toxicity, and antitumor activity of a new type of synthetic compound containing an enediyne functional group capable of benzenoid diradical generation. The design of this cytotoxic molecule was based on the structures of naturally occurring enediyne antibiotics. Compared to the natural compounds, the synthetic enediyne displayed cytotoxicities approaching the natural analogs. Using a tritiated analog, biodistribution studies revealed relatively high uptake levels in kidney, lung, heart, and spleen with moderate levels in all other organs. Antitumor activity was apparent, with significant tumor regression observed in athymic nude mice with established M21 melanomas. Significant tumor antiproliferative effects were observed against L-1210 mouse leukemia, A549 lung carcinomas and PC3 prostate carcinomas in athymic nude mice, and against EMT-6 mouse mammary adenocarcinomas in Balb/cByJ mice. These results suggest that synthetic enediynes may be useful therapeutic compounds since their design reduces systemic toxicity compared to the natural products, without compromising antitumor activity. The relatively low sensitivity of many established cell lines to synthetic enediynes suggests a discrepancy between cell culture and in vivo tumor cytotoxicities. Adaptation of some cell lines for in vivo proliferation may affect their sensitivity to synthetic enediynes.