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A strategy has been devised to physically map replication sites in released chromatin of mammalian cells. When added to the culture medium, 5-bromodeoxyuridine (BrdU) is incorporated into replicating DNA, partially replacing thymidine. BrdU pulses as short as one minute can be visualized on preparations of straightened chromatin fibers from protein-extracted nuclei by means of monoclonal anti-BrdU antibody. Short BrdU pulses (<10 min) appear as strings of fluorescent signals that are 50–300 kb in length. This corresponds to the estimated size of individual replication units. Pulse chase experiments reveal that replicating DNA is tightly associated with the residual nuclear matrix, whereas newly replicated DNA is positioned on the released loop chromatin of nuclear halo preparations. Simultaneous fluorescence in situ hybridization (FISH) on BrdU-substituted released chromatin fibers suggests that replication can initiate at multiple sites anywhere within an α-satellite array of several Mb.