To study the relation between replicon initiation and nuclear organization of DNA, mouse erythroleukemia F4N cells were irradiated with 60Co source and the rates of initiation of DNA synthesis were determined by a sensitive assay based on the introduction of Trioxsalen cross-links in DNA in vivo and determination of the amount of short nascent DNA fragments synthesized between the cross-links. In parallel, nuclear organization of DNA was monitored using the nucleoid sedimentation technique. The results show that DNA initiation rate and relative nucleoid sedimentation change sharply and simultaneously at doses of about 1 Gy, which suggests the existence of relationship between them. This suggestion was supported by the finding, that during the after-irradiation period, first DNA organization was restored and only after this process had been completed, the restoration of replicon initiation commenced. When cells were treated with novobiocin, an agent that is known to slow down the recovery of nucleoid sedimentation rate, initiation of DNA synthesis was also postponed. A hypothesis is put forward that replicon clusters represent groups of adjacent DNA loops organized in superloop domains and that the intact superloop domain structure is necessary for activation of the cluster.