To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-KI cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status by flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTTP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [a-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.It has long been recognized that eucaryotic chromosomes do not replicate as a single element; rather, different sections of chromosomes synthesize their DNA at characteristic times during S phase of the cell cycle, which indicates that each chromosome consists of multiple units of replication (65,66). The existence of subchromosomal replication units in mammalian cells was demonstrated directly, using autoradiography, by Cairns (17) and Huberman and Riggs (42). These experiments also demonstrated that the chronological order of replication of specific DNA segments is invariant from one cell generation to the next (42). Further studies showed that the replication units (replicons) into which chromosomal DNA is organized range in size from 50 to 330 kilobases and appear to initiate in synchronous clusters (25 to 100 replicons per cluster), spaced irregularly along the chromosome and activated in a sequential fashion throughout S pha...