Genetic variation was studied in several mouse L cell lines containing tandemly repeated herpes simplex virus thymidine kinase (TK) genes introduced by DNAmediated gene transfer. Variants were obtained after alternate positive and negative selection for TK expression. Three classes of molecular alteration are described. One class consisted of a concerted wave of hypermethylation affecting many sites in all or nearly all of the TK genes. This resulted in genetically stable TK-variants. Offive TK+ transformants from independent transfer experiments, only one, named HM, showed this class of methylation. Hypermethylation was a reproducible phenomenon in HM, yielding TK-variants after selection with either bromodeoxyuridine or acycloguanosine [Acyclovir or 9-(2-hydroxyethyoxymethyl)guanine]. A second class of alteration consisted of methylation affecting some, but not all, genes in the cluster. This happened in all TK+ (HAT [hypoxanthine-aminopterin-thymidinel-resistant) cell lines investigated, and this second class of methylation was incapable of generating TK-variants. Neither type of methylation was accompanied by genomic rearrangements. The third class of molecular alteration was found among TK+ (HAT-resistant) back revertants of hypermethylated HM TK-derivatives. It consisted of a 10-fold amplification of the hypermethylated TK genes. Demethylation of hypermethylated HM variants was not observed. Thus, hypermethylation in this system can be compensated for by amplification but cannot be reversed.We have used a model cell culture system to explore genetic mechanisms that alter gene expression in eucaryotic cells. A number of mouse L cell variants for expression from a thymidine kinase (TK) gene cluster were characterized in terms of gene copy number, gene arrangement, and DNA modification pattern. Three classes of molecular alteration are described here: two different patterns of DNA methylation, and gene amplification.The tendency of eucaryotic cells to vary phenotypically is an important phenomenon in several biological systems, such as the progressive growth of malignant cells (23), the development of drug resistance in malignant cells (30), the phenotypic variation of cell lines in culture (6,29), and the differentiation of cells during normal development. Among the mechanisms that are known to contribute to this variability are base substitution (1, 6, 29), gene rearrangement (5), and gene amplification (28,32,33,42).DNA methylation has also been broadly implicated in gene regulation, especially for X chromosome inactivation (18,20). A model has been proposed (16,27) in which modification of the DNA sequence to 5-methyl CpG in the recognition sites for regulatory proteins would block gene expression. Heritability of the methylation pattern and the resulting regulatory state would be provided by a maintenance system. After replication of symmetrically methylated CpG sequences, the maintenance system would specifically methylate the new strand in the halfmethylated replication product. Substantial evidence ha...