The lytic-phase origin of DNA replication from human herpesvirus 6B (HHV-6B oriLyt) contains two binding sites for the origin-binding protein (OBPH6B), both of which are required for DNA replication and which are separated by an AT-rich spacer. We have tested the functional significance of the structural, spatial and sequence characteristics of this spacer element by constructing a series of mutated origin sequences and analysing their replication efficiency. Changes in the sequence composition of length of the spacer resulted in dramatic decreases in replication efficiency. Furthermore, in contrast to what has been observed for herpes simplex virus type 1 (HSV-1) oriS, insertion of a complete helical turn of DNA into the spacer also resulted in abrogation of origin function. These data suggest that the arrangement of OBP sites in HHV-6B oriLyt is stringently constrained in terms of spacing and intervening sequence.