Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.