We have analyzed the mechanism of activation of two human ras oncogenes. We have also identified a rasN gene from a human gastric adenocarcinoma which efficiently induced both morphological transformation and tumorigenicity of NIH3T3 cells in a transfection assay. The rasN gene in tumor tissue DNA did not appear to be rearranged or amplified. A molecular clone, which contained an EcoRI fragment spanning the first and second rasN exons, was molecularly cloned directly from the human tumor DNA. Chimeric constructions and DNA sequencing defined the mechanism of activation of the gene as a mutation in the 61st amino acid codon substituting arginine for glutamine. Normal DNA isolated from Epstein-Barr virus immortalized lymphocytes derived from the same patient did not induce morphological transformation or tumorigenicity in NIH3T3 cells. A cloned cell line isolated from the human pancreatic carcinoma cell line Panc1 had previously been shown to contain an activated rasK-2. Sequence analysis of the cloned transfected gene reveals a G to A change within codon 12, which is presumably responsible for its biological activity. This represents the first identification of a codon 12 aspartic acid substitution of a c-rask oncogene from a human tumor-derived cell line.