We have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) containing a 500-fold amplification of a 135-kilobase chromosomal DNA sequence. This sequence includes the gene for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3). The high copy number of the amplified sequence permits it to be visualized as a distinct series of restriction fragments in genomic digests separated on ethidium bromide-stained agarose gels. Initiation of DNA replication in the amplified sequence was studied by radiolabeling DNA synthesized during the onset of S phase in synchronized CHOC 400 cells. Autoradiography of Southern blots of labeled genomic digests shows that DNA synthesis initiates in a small subset of the EcoRI fragments derived from the amplified units. These early labeled fragments are not synthesized at later times during S phase, when different subsets of fragments are synthesized. Regardless of the drug used to collect cells at the beginning of S phase, the replication pattern observed remains the same. These data suggest that replication of the amplified sequence initiates at specific sites within each repeated unit and proceeds in nonrandom order throughout the remainder of the sequence--i.e., that initiation of DNA synthesis in the chromosomes of mammalian cells is sequence specific.