The herpes simplex virus type 1 (HSV-1) origin-binding protein (OBP) is a sequence-specific DNA binding protein encoded by gene UL9 which interacts with the viral origins of DNA replication and also exhibits DNA helicase activity. Sequence-specific DNA binding activity has previously been shown to reside within the C-terminal 317 amino acids, and when expressed alone, this domain exerts a dominant inhibitory effect on HSV-1 DNA synthesis. We have tested several UL9 gene mutants for ability to support or interfere with viral DNA replication. Mutants affected in an ATP binding motif presumed to be associated with DNA helicase activity (ATP-), or defective in origin binding (OBP-) were unable to support replication in a transient assay for HSV-1 origin-dependent DNA synthesis. When the products were screened for their ability to interfere with replication, the ATP- but not the OBP- mutant was inhibitory. Introduction of a mutation which abolished origin-binding activity into the isolated C-terminal fragment also removed the ability to interfere. The C-terminal fragment retained inhibitory activity when the wild-type (wt) protein was specified by a plasmid in which an OBP recognition site within the UL9 gene coding region had been mutated so as to prevent binding without affecting the encoded amino acids. These results suggest that in this assay inhibition of DNA synthesis probably results primarily from competition between mutant and wt forms of OBP for binding to the viral replication origins. The infectivity of HSV-1 DNA in co-transfection experiments was greatly reduced by mutant UL9 proteins which interfered with origin-dependent DNA replication and also by high level expression of the wt polypeptide.