Replication in cellular replicons of mouse Ehrlich ascites, human CCRF-CEM and hamster BHK-21 cells was analyzed, after exposition of the cells to staurosporine, by measuring the overall DNA synthesis rate, by alkaline sedimentation analysis of length distributions of growing daughter strand DNA and by DNA fibre autoradiography. The results consistently indicated that micromolar concentrations of staurosporine caused, in all three cell lines, a fast suppression of replicon initiation which was reversible if the drug treatment did not exceed about 2 h. The inhibition of initiation was accompanied by a slight reduction of rates of propagation of replication forks. The data are interpreted in terms of the existence of a so far unknown factor which seems to be involved relatively directly in the initiation process of cellular replicons and has to be activated, like the large T antigen of SV 40 for the replication initiation in the viral genome, by a specific phosphorylation event. Unlike several other protein phosphorylations of cellular regulation, the kinase concerned here seems to be inhibited only by relatively high staurosporine concentrations.