Flat revertants with reduced malignancy in vivo can be isolated from Kirsten sarcoma virus-transformed NIH3T3 cells following transfection with a normal human fibroblast cDNA expression library. We have recovered from one such revertant a 1.8 kb cDNA clone, "Krev-1", that possesses revertant-inducing activity in transfection assay. The Krev-1 cDNA has the capacity to encode a protein with a calculated molecular weight of 21,000 having strong structural similarity to ras proteins (ca. 50% homology), especially in their GTP/GDP-binding, effector-binding and membrane attachment domains. Point-mutations of Krev-1 protein at the 12th and the 59th amino acid positions from the aminoterminus potentiated the transformation suppressor activity by 2-5 times, and point-mutations within the putative effector-binding domain and at the C-terminal acylation site reduced the activity.