Human insulinoma and renal cell carcinoma DNAs were analysed by a sib-selection tumorigenesis assay to detect the presence of dominant acting oncogenes. Although no novel oncogenes were detected in the five tumor DNAs tested, the increased sensitivity of the tumorigenesis assay allowed detection of overexpressed ras proto-oncogenes activated during transfection. Tumorigenic overexpression of ras proteins occured by two different mechanisms: gene amplification, and increased translation efficiency of a fusion protein. Thirty to fifty fold amplification of a human N-ras gene was detected in primary and secondary mouse tumors. All tumors containing amplified copies of N-ras overexpressed p21 and the cloned gene had no coding sequence mutations. Since DNA from the original human tumor and several metastases did not show N-ras gene amplification, it was concluded that amplification had occured during the primary tumorigenesis assay. In another series of tumors co-integration of the mouse c-H -ras gene during the secondary transfection resulted in tumorigenic overexpression of ras protein without elevation of mRNA levels. Three c-H-ras cDNA clones from secondary tumor RNA contained no coding sequence mutations, but were divergent at the 3'end. Polymerase chain reaction amplification of RNA showed novel alternative splicing at intron E of mouse c-H-ras mRNA in both tumors and untransfected cells. An upstream ATG was identified that potentially initiates translation of an open reading frame (ORF) overlapping the H-ras p21 translation initiation site. A single base deletion within exon -1 of the cDNA clones placed this upstream ATG in frame with the ras coding sequence, creating a potential fusion protein. Translation of this mRNA in rabbit reticulocyte extracts and Xenopus oocytes showed exclusive production of a p23 ras fusion protein. When the upstream ATG was deleted, only p21 ras was translated in both systems. Based on these results it is proposed that in vivo recognition of the upstream ATG and translation of the ORF overlapping the p21 start site might serve to modulate the translation of p21. The single base deletion and resultant ras fusion protein may constitute a novel mechanism of ras overexpression by circumventing this translational regulation .