A cDNA clone of the dbl transforming gene mRNA was isolated and sequenced. Computer analysis of the predicted dbl protein indicated it to be highly hydrophilic with no hydrophobic domain characteristic of a membrane-spanning region or signal peptide. cDNA clones representing the human dbl proto-oncogene were also isolated. Nucleotide sequence analysis indicated that a stretch of 300 amino acids within the N-terminal half of proto-dbl showed structural similarity to the intermediate filament vimentin. Under the influence of the same strong promoter, both genes would readily transform NIH/3T3 cells, but the dbl oncogene cDNA exhibited higher transforming capability. The expression of the dbl oncogene in human normal tissues and childhood tumors of neuroectodermal origin was investigated. Proto-dbl RNA species were detected only in fetal brain and adrenal fluids as well as in adult tests and ovaries. Moreover, the dbl oncogene was found to be transcribed in Ewing,s sarcoma as a single 5.3-kb pi mRNA species, while it was not present in two related categories of tumors, neuroblastoma and neuroepithelioma. Using peptide antisera raised against specific dbl peptides, the proto-oncogene product was identified as a 115- kDa protein, phosphorylated in serine and threonine, and localized in the crude membrane and soluble fraction of the cell. DNAs from 14 of 33 mouse liver tumors induced by a single injection of N-nitrosodiethylamine (DEN) at 12-15 days of age were able to induce morphological transformation of NIH/3T3 cells. Analysis of the transformants obtained revealed the presence of activating mutations in the H-ras gene.