The mechanism for induction of tumors by estrogens is still unresolved. Neoplastic transformation of Syrian hamster embryo fibroblasts by diethylstilbestrol (DES) suggests that established principles of chemical carcinogenesis may be involved. The Syrian hamster embryo fibroblast cells provide a system in which now the question can be asked whether the metabolic activation of DES is a prerequisite for its biological activity in this system. In this study, Syrian hamster embryo fibroblast cell cultures were shown to oxidatively metabolize DES to cis,cisdienestrol (Z,Z-DIES) which is a DES metabolite commonly found in vivo. The only other metabolic conversion of DES detectable in these cell cultures was the formation of the glucuronides of DES and Z,Z-DIES. Z,Z-DIES is formed more efficiently in incubations with rapidly growing cells than in cultures approaching confluence. When arachidonic acid was added to the medium, Z,Z-DIES formation was enhanced, whereas indomethacin added to the cell cultures inhibited the formation of this metabolite. These data suggest the involvement of prostaglandin synthetase in the oxidative metabolism of DES by Syrian hamster embryo fibroblast cells in culture and suggest that cooxidation may play a role for its biotransformation in whole cells. Moreover, since many competing metabolic pathways are available to DES in vivo, this present study adds important additional support to the hypothesis that metabolism of DES via a peroxidative route plays a role in its carcinogenicity.