When human cells enter S-phase, overlapping differential inhibitory mechanisms downregulate the replication licensing factors ORC1, CDC6 and Cdt1. Such regulation prevents re-replication so that deregulation of any individual factor alone would not be expected to induce overt re-replication. However, this has been challenged by the fact that overexpression of Cdt1 or Cdt1+CDC6 causes re-replication in some cancer cell lines. We thought it important to analyze licensing regulations in human non-cancerous cells that are resistant to Cdt1-induced re-replication and examined whether simultaneous deregulation of these licensing factors induces re-replication in two such cell lines, including human fibroblasts immortalized by telomerase. Individual overexpression of either Cdt1, ORC1 or CDC6 induced no detectable re-replication. However, with Cdt1+ORC1 or Cdt1+CDC6, some re-replication was detectable and coexpression of Cdt1+ORC1+CDC6 synergistically acted to give strong re-replication with increased mini-chromosome maintenance (MCM) loading. Coexpression of ORC1+CDC6 was without effect. These results suggest that, although Cdt1 regulation is the key step, differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells. Our findings also show for the first time the importance of ORC1 regulation for prevention of re-replication.