H-Tyr329-Ala330-Gly331-Ala332-Val333-Va l334-Asn335-Asp336-Leu337-OH, the C-terminal end of herpes simplex virus ribonucleotide reductase subunit 2 (HSV R2), specifically inhibits viral enzyme activity by interacting with subunit 1 (HSV R1). In a previous structure-activity study, we identified four sites on the nonapeptide where the inhibitory potency could be modulated: a minimum active core 333-337, a spacer segment 330-332, and the N- and C-termini. To further explore the structural features of HSV R2-(329-337) that are required to obtain a potent inhibition, a series of analogues comprising modifications in these four regions were synthesized by solid-phase methodology. Changes in the segment 333-337 of the molecule decreased the inhibitory potency by more than 2-fold, except for the Ile334 substitution, which resulted in a 1.5-fold increase in potency. Replacement of Tyr329 by other aromatic or aliphatic amino acids diminished the nonapeptide activity from 1.4-fold to 5.9-fold. The spacer segment contributed to enhance potency. Modification with amino acids that could induce conformational changes, such as Pro or D-Ala, generated compounds with a similar or lower activity, respectively. Amidation or amino acyl addition at the carboxylic end was detrimental while acylation of the N-terminus was generally beneficial for the inhibitory potency. Disubstitution in position 332 and 334 by Thr and Ile, which are present in the C-terminal portion of varicella-zoster virus ribonucleotide reductase subunit 2, resulted in a peptide that is 4.0 times more potent than HSV R2-(329-337), while each monosubstitution alone generated peptides with 150% of the activity of HSV R2-(329-337) nonapeptide. These results indicate a synergistic effect of the disubstitution which confers to this analogue physicochemical properties enhancing its ability to interact with its R1 binding site.