Normal human cells in culture have never been neoplastically transformed by carcinogen exposure. One possible explanation is that the life span of such cells is too short for them to acquire the necessary changes. To test this hypothesis, we needed normal human cells with a greatly extended or an infinite life span. We transfected the v-myc gene and a selectable marker into normal human fibroblasts, identified a drug-resistant clone expressing v-myc protein, and passaged the progeny of the clone until they senesced. A few cells continued to proliferate and gave rise to a diploid, infinite life span cell strain, MSU-1.0, that has normal growth control and is nontumorigenic in athymic mice. Analysis showed that one more genetic change, in addition to unregulated expression of the v-myc gene, was involved in generating MSU-1.0 cells. They spontaneously gave rise to a variant strain, designated MSU-1.1, that grows more rapidly and is less dependent on exogenous growth factors. Analysis showed that at least two additional changes were involved in generating this cell strain. It has a stable karyotype composed of 45 chromosomes, including two markers. Transfection of specific oncogenes was used to determine the number and nature of additional changes required to transform MSU-1.1 cells into malignant cells. Analysis indicated that two changes were involved, but no change in karyotype. Exposure of MSU-1.1 cells to a single carcinogen treatment, followed by selection for cells with the characteristics of oncogene-transformed MSU-1.1 cells also yielded malignant human cells. We conclude that malignant transformation of normal human fibroblasts requires six or seven genetic changes, some of which involve suppressor genes.