The origin recognition complex (ORC) is involved in formation of prereplicative complexes (pre-RCs) on replication origins in the G1 phase. At the G1/S transition, elevated cyclin E-CDK2 activity triggers DNA replication to enter S phase. The CDK cycle works as an engine that drives progression of cell cycle events by successive activation of different types of cyclin-CDK. However, how the CDK cycle is coordinated with replication initiation remains elusive. Here we report that acute depletion of ORC2 by RNA interference (RNAi) arrests cells with low cyclin E-CDK2 activity. This result suggests that loss of a replication initiation protein prevents progression of the CDK cycle in G1. p27 and p21 proteins accumulate following ORC2 RNAi and are required for the CDK2 inhibition. Restoration of CDK activity by co-depletion of p27 and p21 allows many ORC2-depleted cells to enter S phase and go on to mitosis. However, in some cells the release of the CDK2 block caused catastrophic events like apoptosis. Therefore, the CDK2 inhibition observed following ORC2 RNAi seems to protect cells from premature S phase entry and crisis in DNA replication. These results demonstrate an unexpected role of ORC2 in CDK2 activation, a linkage that could be important for maintaining genomic stability. © 2005 by The American Society for Biochemistry and Molecular Biology. Inc.