Patterns of methylation of CpG dinucleotides in the promoter region of the thymidine kinase (TK) gene in wild-type and TK-deficient Chinese hamster cell lines were studied. Whereas wild-type cells were unmethylated, three conventionally derived TK-deficient cell lines were all almost completely methylated in the promoter region. Demethylation at a number of different CpG sites was observed upon selection for reexpression of the TK gene. Of thirteen HhaI (GCGC) or HpaII (CCGG) sites studied, the highest correlation between absence of methylation and at least partial TK activity was obtained at one HhaI site within 20 bp of the putative cap site. Silencing in the three conventionally derived mutants is therefore accompanied by hypermethylation of the promoter-associated CpG-rich island. We contrast this situation with another type of silencing event, in which TK was coordinately inactivated at a high frequency with at least one other linked allele. Methylation of the promoter region of TK was not associated with this event, but two lines of evidence suggested a role for methylation at sites other than in the promoter region of TK.