Neisseria gonorrhoeae produces several antioxidant defenses, including high levels of catalase, which may facilitate the persistence during an inflammatory response via neutralization of H2O2 produced by phagocytes. In vivo testing of the role of catalase in gonococcal survival is critical since several physiological factors impact interactions between N. gonorrhoeae and polymorphonuclear leukocytes (PMNs). Here we assessed the importance of gonococcal catalase in a surrogate model of female genital tract infection. Female BALB/c mice were treated with 17-beta estradiol to promote susceptibility to N. gonorrhoeae and inoculated intravaginally with wild-type gonococci or a catalase (kat) deletion mutant. A localized PMN influx occurred in an average of 43 and 81% of mice infected with wild-type or kat mutant gonococci, respectively, and PMNs associated with numerous wild-type or catalase-deficient bacteria were observed in vaginal smears. The combined results of six experiments showed a significant difference in the number of days wild-type bacteria were recovered compared to the catalase-deficient gonococci. However, there was much variability between experiments, and we found no correlation between PMN influx, colonization load, and clearance of wild-type or kat mutant bacteria. Estradiol treatment did not impair bacterial uptake, the luminol-dependent chemiluminescence response, or the killing capacity of isolated murine PMNs against N. gonorrhoeae or Staphylococcus aureus. Our data suggest N. gonorrhoeae is not significantly challenged by H2O2 produced by PMNs in the murine lower genital tract; alternatively, redundant defense mechanisms may protect the gonococcus from reactive oxygen species during infection.