The transforming activity of luteoskyrin (LS), a bis-anthraquinoid mycotoxin produced by Penicillium islandicum Sopp., and a hepatocarcinogen in rodents, was examined by an in vitro transformation assay using mouse embryonal Balb/3T3 A31-1-1 cells. The results revealed that LS induced type III foci at 0.5 micrograms/ml, and that the cells selected from these foci by soft-agar cloning grew with a high saturation density. Thus, it was confirmed that LS not only induces hepatic tumours in laboratory animals, but also transforms in vitro cultured mammalian cells. The tumorigenicity of the transformants obtained was confirmed by transplantation into nude mice and by image analysis with IIIIn. A transfection assay, using calcium phosphate co-precipitation, demonstrated that the DNA of the cloned cells transformed NIH3T3 cells. Northern blot also revealed transcriptional activation of c-myc and c-Ha-ras oncogenes. The possible participation of LS-derived hydroxy radicals in the formation of genetic lesions was discussed.