We report here comparative studies of DNA methylation and expression of intracisternal A-particle (IAP) genes in murine plasmacytomas, TEPC-15 and MOPC-315, in NIH/3T3 embryo fibroblasts, and in normal liver from young adult BALB/c mice. In Southern blot analysis using methylation-sensitive restriction enzymes, we observed hypomethylation of 4.7- and 5.3-kilobase pair IAP proviruses, and partial undermethylation of other IAP DNA fragments in TEPC-15, MOPC-315, and 3T3 cells, while most of these DNA fragments were hypermethylated in liver. The extent of undermethylation at 12 different sites including the long terminal repeat sequence region and 5′-flanking sequence within one specific IAP gene was found to be similar between 3T3 cells and plasmacytomas. Little or no undermethylation of these sites was found in liver. Hypomethylation of IAP genes, however, is not correlated with their expression in 3T3 cells (as it is found for plasmacytomas), since 3T3 cells, like liver cells, have no detectable transcripts. We also observed hypomethylation of the k-light chain gene in 3T3 cells, suggesting that undermethylation may be a generalized phenomenon in these cells. The relationships between gene undermethylation and the control of gene expression in 3T3 cells is discussed.