The origin-binding protein (OBP) from herpes simplex virus 1 is a member of the SF2 helicase superfamily and is required for the initiation of DNA synthesis from a viral origin of DNA replication (oriS). The high-affinity binding sites for OBP in oriS, boxes I and II, are separated by an A+T-rich spacer. We used the gel retardation technique to examine the influence of this spacer sequence on the formation of a specific complex, referred to as complex II, between OBP and oriS. The formation of this OBP-oriS complex was greatly promoted by adenosine 5'-[gamma-thio]triphosphate and other nucleotide cofactors. Surprisingly, oriS constructs where the spacer sequence had been altered with approximately half of a helical turn (+4 or -6 base pairs) supported the formation of a more stable complex II than the wild-type origin. DNase I footprinting experiments showed that the cooperative binding of OBP to boxes I and II was affected by the length of the spacer sequence in the same way. In contrast, the ability of oriS-containing plasmids to replicate was most efficient with wild-type oriS. This paradox can be resolved if it is assumed that an ATP-dependent cooperative binding of OBP to properly spaced recognition sequences in oriS is required to induce a conformational change of DNA, thereby facilitating initiation of DNA replication.