ORIGINS Of DNA replication in Saccharomyces cerevisiae are bound by two protein complexes during the cell cycle(1,2). Post-replicative complexes closely resemble those generated in vitro by purified origin recognition complex (ORC)(1,3-5), which is essential for DNA replication in vivo(6-11). Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuclease protection overlapping the ORC footprint(1). We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo(12-16), is essential for the establishment and maintenance of pre-RCs, suggesting that it is a component of these complexes. Without Cdc6p, G1 origins closely resemble post-replicative origins, providing evidence that ORC is also a component of pre-RCs, These results suggest that pre-RCs play an essential role in initiating DNA replication and support a two-step mechanism for the assembly of functional initiation complexes.