The relationship between replication origin/termination sites and nuclear matrix was investigated by DNase-digestion of the halo structure in synchronized LP1-1 cells. The LP1-1 cells, which were arrested in the G₁ /S boundary by serum starvation and hydroxyurea treatment, entered into S phase upon the readdition of fresh medium. The parental DNA remaining on the nuclear matrix isolated from the S_0 stage cells decreased while the ratio of radioactivity (³H/^(14)C) on the nuclear matrix increased, with DNase digestion time, suggesting that the replication origins are associated with the nuclear matrix and that DNA replication occurs at the nuclear matrix. The DNA fragments pulse-labeled at S_0. 3C-pause, and S_8 stages were more resistant to DNase I digestion than those for the other S stages. Similar results were obtained from the pulse-chase experiments, in which pulse-labeling performed at each stage of S phase was followed by further incubation for 1 hr. These results suggest that the replication origins and termination sites may be fixed at nuclear matrix, and that the fragments pulse-labeled at 3C-pause may contain a large population of replication origins and/or termination sites. Each replicon, therefore, could be expected to form a double loop by matrix-binding of DNA at three points with one origin and two termination sites, as proposed by Carri et al. (Exp. Cell Res. 164, 426-436 (1986)).