To determine if expression of mutant p21 ras could convert Simian Virus 40-immortalized human uroepithelial cell line (SV-HUC) to tumorigenicity, SV-HUC cells were transfected with pSV2-neo (a neomycin-resistant gene) or PREJ/ras (c-HA-ras-1 with the 12th codon mutation and neo). Seven independent G418-resistant clones (A----G) were isolated from each group (SV-HUC/ras and SV-HUC/neo). SV-HUC/ras clones were morphologically altered, while SV-HUC/neo clones retained a typical SV-HUC epithelial morphology. Electrophoretic analysis of immunoprecipitated ras proteins detected altered p21 ras protein in four of seven SV-HUC/ras clones at passage (P)2 and in five of seven clones at P12 posttransfection. The relative levels of ras p21 differed among the clones and appeared to increase with passage in culture. RNA and DNA dot blot analyses showed that clones with more abundant mutant p21 also had higher ras RNA levels and, in one case, increased ras gene copy number. No altered ras protein was detected in any SV-HUC/neo clones. ras- and neo-transfected clones were tested for tumorigenicity at P2 posttransfection and again at P12 by four s.c. inoculations each into athymic nude mice. None of 56 inoculations of SV-HUC/neo clones was tumorigenic. None of the SV-HUC/ras clones at P2 gave rise to tumors at all four injection sites. However, two ras-transfected clones, SV-HUC/ras-B and SV-HUC/ras-F, produced one tumor each. One clone, SV-HUC/ras-D which produced abundant mutant p21, was negative when inoculated at P2, but produced tumors in four of four sites when reinoculated after ten passages in vitro. All tumorigenic clones had detectable levels of mutant ras p21. However, the relative levels of altered p21 ras protein among the SV-HUC/ras clones did not directly predict their tumorigenic potential, as several nontumorigenic SV-HUC/ras clones had protein levels equal to or higher than the most tumorigenic clone (SV-HUC/ras-D at P12). Cell lines established from the tumor explants exhibited higher ras gene copy numbers, higher RNA levels, and more abundant p21 than was seen in the clones at the time of inoculation. Therefore, increases in ras protein abundance occurred during tumor formation in vivo, as well as during passage of cells in culture, and such cells apparently had a selective growth advantage. However, expression of abundant mutant ras protein was not in itself sufficient for neoplastic transformation of SV-HUC.(ABSTRACT TRUNCATED AT 400 WORDS)