A flat revertant, Rl, was isolated from humgn activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. Rl contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from Rl cells but Rl cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that RI was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in Rl is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.It has been found that 10 to 20% of human tumors have a mutation in one of the three ras oncogenes (Ha-ras, Ki-ras and N-ras) leading to the production of p21 ras oncoproteins, which are thought to play an important role in the transformed phenotype (1). The ras proteins bind GTP and GDP and have intrinsic GTPase activity. They may control cell proliferation by regulating a signal transduction pathway as do the regulatory G proteins (1). However, the biochemical mode of action and the biological target molecules of the ras proteins are unknown. Recently, the activating mutations of the ras genes have been detected not only in malignant tumors such as colorectal tumors but also in benign tumors such as colon adenomas (3, 9). The former showed somatic loss of chromosome sequences, but the latter did not (8). Such chromosomal changes as monosomy or trisomy were also described in hamster cells transfected with v-Ha-ras or human activated c-Ha-ras-1 (hu-ac-Ha-ras) genes (23,31). All of these findings suggest that ras activation is not sufficient and that alteration of cellular transformation suppressor genes is necessary for malignant transformation of cells. However, little is known about the suppressor genes or their products. Isolation of mutant cells that contain alterations in one or more of the suppressor genes involved in transformation by oncogenes could be one approach to this problem.The EJ-NIH 3T3 cell line, which is an NIH 3T3 cell line carrying the transfected hu-ac-Ha-ras sequence of EJ human bladder carcinoma cells, kindly provided by T. Y. Shih (National Cancer Institute, Frederick, Md.; 34) showed the typical morphology of transformed cells. EJ-NIH 3T3 cells (106) were treated with ethyl methanesulfonate (200 ,ug/ml for 24 h) and 8-azaguanine (5 ,ug/ml). Two mutant clones, Rl and R2, were selected from the population. They showed a flat, typical spindle shape and an ordered growth pattern of fibroblast or endothelial cells. They had lost the ability to pile up at random and became contact inhibited ...