The influence of c-myc expression on fibroblast growth and morphology was investigated by transfection of c-myc genes linked to viral promoters. No foci were observed after transfection of either NIH/3T3 or Rat 2 cells. Cell lines containing activated c-myc genes were established using SV2-neo coselection and several growth parameters of the cells were studied. The cells showed a slight increase in refractility and formed colonies in soft agar with an efficiency of only 1%-2%. The c-myc-transfected cells grew well in 0.5% serum while the controls did not. The major difference in cell growth noted was that c-myc-transfected cells were tumorigenic when inoculated into nude mice or syngeneic rats. Analysis of RNA from the tumorigenic cells showed a level of c-myc expression from the transfected genes that was 2 to 6 fold higher than that from the endogenous gene. The level of c-myc RNA in the fibroblast tumors was similar to that found in mouse plasmacytomas. Expression of the endogenous c-myc gene was unaffected by the transfected genes for subconfluent cells in culture, but the gene was shut off in the nude mouse tumors. These results demonstrate that constitutive c-myc expression leads to tumorigenicity in immortalized cell lines.