Eukaryotic DNA replication requires the assembly of multiprotein pre-replication complexes (pre-RCs) at chromosomal origins of DNA replication. Here we describe the interactions of highly purified Schizosaccharomyces pombe pre-RC components, SpORC, SpCdc18, and SpCdt1, with each other and with ars1 origin DNA. We show that SpORC binds DNA in at least two steps. The first step likely involves electrostatic interactions between the AT-hook motifs of SpOrc4 and AT tracts in ars1 DNA and results in the formation of a salt-sensitive complex. In the second step, the salt-sensitive complex is slowly converted to a salt-stable complex that involves additional interactions between SpORC and DNA. Binding of SpORC to ars1 DNA is facilitated by negative supercoiling and is accompanied by changes in DNA topology, suggesting that SpORC-DNA complexes contain underwound or negatively writhed DNA. Purified human origin recognition complex (ORC) induces similar topological changes in origin DNA, indicating that this property of ORC is conserved in eukaryotic evolution and plays an important role in ORC function. We also show that SpCdc18 and SpCdt1 form a binary complex that has greater affinity for DNA than either protein alone. In addition, both proteins contribute significantly to the stability of the initial SpORC-DNA complex and enhance the SpORC-dependent topology changes in origin DNA. Thus, the formation of stable protein-DNA complexes at S. pombe origins of replication involves binary interactions among all three proteins, as well as interactions of both SpORC and SpCdt1-SpCdc18 with origin DNA. These findings demonstrate that SpORC is not the sole determinant of origin recognition.