Malignant transformation of cells in vitro requires the action of cooperating oncogenes. However, the action of a single activated oncogene, if placed under a strong constitutive promoter, is sufficient to transform in vitro established cells. We have investigated the role of cooperating oncogenes in the transformation of normal rat bladder epithelial cells. Effects of polyoma middle T, v-Ha-ras or activated rat c-Ha-ras, independently or in combination with v-myc on in vitro and in vivo behavior of rat bladder epithelial cells were studied. Introduction of polyoma middle T alone, c-Ha-ras or in some cases v-myc into "normal" rat bladder epithelial cells was sufficient for full malignant transformation of these cells. In addition, introduction of polyoma middle T, activated c-Ha-ras or v-myc transformed cells into nude mice, resulted in development of highly invasive adenocarcinomas. These results indicate that full malignant transformation of normal rat bladder epithelial cells did not require the action of introduced cooperating oncogenes.