The cause of deeply invasive human bladder carcinoma is unknown. Animal studies suggest that a malignant (invasive) conversion is inducible in low-grade noninvasive tumors by further exposure to a chemical carcinogen. To elucidate what molecular mechanism(s) is involved in the conversion, an in vitro system has been established in which conversion from low- to high-grade carcinoma can be induced. A rat bladder carcinoma cell line D44, derived from an N-methyl-N-nitrosourea (MNU)-induced low-grade noninvasive rat bladder carcinoma was used in the present investigation. Cloned D44 cells (D44c) were exposed to MNU, 50 to 400 micrograms/ml, for 1 h at 37 degrees C once a week for up to 6 weeks. After exposure to MNU, cells with altered morphology were cloned. The yield of altered clones was highest after a total dose of 150 to 200 micrograms of MNU used in 1 to 3 doses. Of 21 clones with altered morphology, 4 clones were further treated with MNU at the initial dose once a week for up to 3 weeks and then subcloned. Thirty-three of these subclones were examined for tumorigenicity in athymic nude mice. Twenty-seven formed highly invasive carcinomas, mostly squamous type, whereas the parental D44c cells failed to develop tumors upon inoculation. Pulmonary metastases were observed in 17 of the 27 clones. Plasminogen activator activity was elevated 4- to 9-fold as compared to parent D44c cells. ras p21 mutations at codon 12 were detected in 5 of 30 clones. These results indicate that the in vitro system described here may provide a useful model to study the molecular mechanisms involved in the conversion of noninvasive bladder carcinomas to metastasizing ones.