Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, on5S1, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-P, is -17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oris14 is 127 bp long, and the DMI in ori-is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMImediated regulation of mammalian chromosomal origins are considered.To investigate the relationship between DNA cytosine methylation and site-specific point mutations in mammalian genes, we analyzed the positions of cytosine methylation in genomic DNA encoding the cloned Chinese hamster ovary (CHO) cell gene for ribosomal protein S14 (RPS14) and compared the methylation sites detected with the position of a recurrent transition mutation affecting the gene's fifth exon (39). During the course of that study, we noted an unusual methylation pattern within a short stretch of the DNA sequence at the 3' end of the gene. Although cytosine methylation at the 5' end and middle of CHO RPS14 occurs exclusively at CpG dinucleotides, in exponentially growing cells actively engaged in DNA replication, we found a unique chromosome segment at the 3' end of the gene in which every dC residue was methylated. Within this densely methylated island (DMI), both DNA strands were fully methylated, and 5-methyl cytosines were detected in CpA, CpT, and CpC as well as CpG dinucleotides.In the accompanying paper, we report that the 3' end of the CHO RPS14 locus also encodes an early-S-phase origin of bidirectional chromosomal DNA replication (OBR) designated oiS14 (40