During experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere. Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than in fibroblasts and that a triple dose of one or more genes may be the initial event in this process.