We have designed an assay to measure the rate of initiation of DNA synthesis in Friend erythroleukemia cells and have shown that this parameter is reduced by gamma-radiation and treatment with 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylene-beta-D-glucopyranoside) (VP-16). It is concluded, that double-strand breaks in DNA are the immediate cause for this effect. The decrease in the rate of replicon initiation is affected differently by different agents such as cis-diamminedichloroplatinum(II), cycloheximide, staurosporine, and 3-aminobenzamide. The analysis of these results indicates that the observed partial decrease of the rate of DNA initiation is most probably transmitted from the site of damage to the initiation site by one or more phosphorylation/dephosphorylation steps. It does not require de novo synthesis of protein factors, but is probably dependent on poly(ADP-ribosyl)ation of chromatin at the site of DNA breaks.