Origins of DNA replication in Saccharomyces cerevisiae are bound by two protein complexes during the cell cycle. Post-replicative complexes closely resemble those generated in vitro by purified origin recognition complex (ORC), which is essential for DNA replication in vivo. Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuclease protection overlapping the ORC footprint. We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo, 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.