A naturally occurring population of polymorphonuclear leukocytes (PMNs) was detected in the vaginal lumen of uninoculated mice. A large population of these cells also infiltrated the vaginal mucosa following intravaginal HSV-2 inoculation. We examined the role of PMNs in preventing infection of the vaginal mucosa, virus clearance, and limiting virus spread to the lumbosacral ganglia. Depletion of PMNs prior to HSV-2 inoculation did not increase the incidence of infection suggesting that the small population of resident PMNs was ineffective in preventing infection by a viral pathogen. Depletion of PMNs impacted virus clearance from the vagina over a range of HSV-2 doses resulting in significantly higher virus titers on days 4 through 6 after inoculation. Virus clearance was delayed in PMN-depleted immune mu MT mice suggesting that PMN involvement in HSV-2 clearance did not require specific antibody. PMN-depletion of non-immune mice increased virus spread to the sensory ganglia only in mice inoculated with high virus doses. Immunization of mice with an attenuated strain of HSV-2 protected the sensory ganglia against acute infection with a challenge strain. Although PMN depletion of immune mice significantly increased virus titers in the vagina, the incidence of acute virus replication in the sensory ganglia was not different than in control-treated immune mice suggesting that PMNs were not required for protection of the sensory ganglia in immune animals. Taken together, these results suggest that PMNs were involved in resolving genital HSV-2 infections, but played only a limited role in preventing HSV-2 spread to the sensory ganglia.