Herpes simplex virus (HSV) encodes its own ribonucleotide reductase (RR), which provides the high levels of deoxynucleoside triphosphates required for viral DNA replication in infected cells. HSV RR is composed of two distinct subunits, R1 and R2, whose association is required for enzymatic activity. Peptidomimetic inhibitors that mimic the C-terminal amino acids of R2 inhibit HSV RR by preventing the association of R1 and R2. These compounds are candidate antiviral therapeutic agents. Here we describe the in vitro selection of HSV type 1 KOS variants with three- to ninefold-decreased sensitivity to the RR inhibitor BILD 733. The resistant isolates have growth properties in vitro similar to those of wild-type KOS but are more sensitive to acyclovir, possibly as a consequence of functional impairment of their RRs. A single amino acid substitution in R1 (Ala-1091 to Ser) was associated with threefold resistance to BILD 733, whereas an additional substitution (Pro-1090 to Leu) was required for higher levels of resistance. These mutations were reintroduced into HSV type 1 KOS and shown to be sufficient to confer the resistance phenotype. Studies in vitro with RRs isolated from cells infected with these mutant viruses demonstrated that these RRs bind BILD 733 more weakly than the wild-type enzyme and are also functionally impaired, exhibiting an elevated dissociation constant (Kd) for R1-R2 subunit association and/or reduced activity (kcat). This work provides evidence that the C-terminal end of HSV R1 (residues 1090 and 1091) is involved in R2 binding interactions and demonstrates that resistance to subunit association inhibitors may be associated with compromised activity of the target enzyme.