Early passages of the human teratocarcinoma cell line PA1 are not tumorigenic in nude mice, while late passages are. A transforming gene present in late passages of PA1 cells was isolated as a biologically active molecular clone and is a new isolate of the human rasN locus. Its transforming activity is due to a single G → A (G, guanine; A, adenine) point mutation at the codonfor amino acid 12 which changes the codon for glycine so that an aspartic acid residue is expressed. In contrast to late passage PA1 cells (passages 106, 330, and 338), DNA from the PA1 cell line at early passages (passage 36) does not yield rasN foci in DNA transfection assays. Thus, the presence of an activated rasN in PA1 cells correlates with enhanced tumorigenicity of the cell line and, more importantly, may have arisen during cell culture in vitro.