A two-stage transformation protocol was used to chemically transform the mouse embryo fibroblasts, C3H/10T1/2 Cl 8. To initiate the cells 0.37 μM 20-methyicholanthrene was used and 0.17 μM of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate was employed to complete the transformation process. Six weeks later transformed foci were identified and isolated by the ring-cloning technique. Altogether eight different foci were trypsinized resulting in a total of 12 morphologically transformed subclones. Three of these clones, designated TPA 41, TPA 42 and TPA 482, have been characterized in detail. Their growth morphologies were different. The TPA 482 cells grew in a criss-cross pattern with piled up foci, thus showing a characteristic type III morphology. The TPA 482 clone did not show cell-density growth inhibition and grew in soft agar. The TPA 41 and TPA 42 clones exhibited cell-density growth inhibition, grew as monolayers and formed only few colonies in soft agar. Late passages of the TPA 42 clone acquired growth characteristics similar to TPA 482. The C3H/1OT1/2 Cl 8 and the TPA 41 cells were not tumorigenic when transplanted into syngeneic mice. TPA 482 cells were strongly tumorigenic, producing tumors in 6/6 mice in 21 days. The TPA 42 cells were also tumorigenic, the first tumors appearing after 4 weeks; all animals injected with TPA 42 cells had tumors after 8 weeks. All tumors observed appeared to be fibrosarcomas. Flow cytometric analysis indicated differences in DNA distributions between tumor cells grown in vitro and the tumors in vivo. Two-dimensional gel analysis of the total cellular and the nuclear proteins showed an increase in the TPA 42 and TPA 482 cells of an acidic 48 000 and a basic 83 000 mol. wt polypeptides, and a decrease of a neutral polypeptide of mol. wt 46 000, located in the nucleus of TPA 482 cells.