The antibiotic acetomycin was active in vitro against HCT-8 human colon adenocarcinoma cells (IC50, 1.5 microgram/ml) and L1210 murine leukemia cells (IC50, 2.2 micrograms/ml). Acetomycin also had marked activity in the human tumor stem cell assay, with a 33% overall response rate (less than or equal to 30% survival) against 49 primary tumors. However, acetomycin was inactive in four in vivo tumor assay systems (L1210 and P388 leukemias, B16 melanoma and the MX-1 mammary xenograft system). This lack of in vivo activity may result from metabolic inactivation of acetomycin.