We rediscovered anisomycin in the course of our antitumor screening using human tumor cell lines. Here we report that anisomycin and some new relatives (named 3097-compounds) were isolated as antitumor substances showing cytotoxicity against human tumor cell lines in vitro. The active substances were obtained from a fermentation broth of Streptomyces strain SA3097 isolated from a soil sample collected from a sub-marine deposit on the coast of Miura City, Kanagawa, Japan. Six compounds were purified, among which three were known: C1 was anisomycin, D1 was deacetylanisomycin, and D2 was 2-(p-hydroxyphenyl)methyl-3,4-dihydroxypyrrolidine (deacetyldemethylanisomycin); the rest were new congeners of anisomycin. The activity of the three new congeners against two human tumor cell lines (LU99: human lung carcinoma; MCF7: human breast cancer) was tested. Structure-activity relationship analysis showed that acylation of the pyrrolidine ring was important for cytotoxicity; deacylation of the pyrrolidine ring (D1) resulted in considerable reduction of activity, and additional demethylation in the aromatic ring (D2) caused complete loss of activity. This is consistent with the mechanism of action of anisomycin, which involves inhibition of protein synthesis, as deacetyldemethylanisomycin was previously found to have far lower activity than anisomycin in a cell-free protein synthesis system.