Comparative studies of DNA isolated from adult C3H mouse liver, immortalized mouse embryo cells (C3H/10T1/2 Cl 8) and the tumorigenic methylcholanthrene transformed C3H/10T1/2 Cl 16 cell line have been carried out in order to analyze possible structural changes in the ribosomal genes associated with the immortalization and tumorigenic transformation of mouse cells. Southern blot hybridization experiments revealed a mutation hotspot within repetitive sequences 13 kb upstream from the 18S rRNA genes in the non-transcribed spacer (NTS). Other DNA changes were localized near the initiation and termination regions of rRNA transcription. The differences found in the restriction maps of the 5'-region resided 5 to 6 kd upstream from the 18S 5'-end and the changes located in the 3'-end mapped approximately 5 kb downstream from the 28S 3'-end. Thus, oncogenic transformation of the C3H/10T1/2 Cl 8 cells by methylcholanthrene treatment was associated with stable genetic changes in the 18S rRNA gene. There was no evidence for rRNA gene amplification.