ABSTRACTChlamydia trachomatisandNeisseria gonorrhoeaeare the most frequently reported agents of bacterial sexually transmitted disease worldwide. Nonetheless,C. trachomatis/N. gonorrhoeaecoinfection remains understudied.C. trachomatis/N. gonorrhoeaecoinfections are more common than expected by chance, suggestingC. trachomatis/N. gonorrhoeaeinteraction, andN. gonorrhoeaeinfection may reactivate genital chlamydial shedding in women with latent (quiescent) chlamydial infection. We hypothesized thatN. gonorrhoeaewould reactivate latent genitalChlamydiamuridaruminfection in mice. Two groups ofC. muridarum-infected mice were allowed to transition into genital latency. One group was then vaginally inoculated withN. gonorrhoeae; a third group receivedN. gonorrhoeaealone.C. muridarumandN. gonorrhoeaevaginal shedding was measured over time in the coinfected and singly infected groups. ViableC. muridarumwas absent from vaginal swabs but detected in rectal swabs, confirmingC. muridarumgenital latency and consistent with the intestinal tract as aC. muridarumreservoir.C. muridaruminclusions were observed in large intestinal, but not genital, tissues during latency. Oviduct dilation was associated withC. muridaruminfection, as expected. Contradicting our hypothesis,N. gonorrhoeaecoinfection did not reactivate latentC. muridarumvaginal shedding. In addition, latentC. muridaruminfection did not modulate recovery of vaginal viableN. gonorrhoeae. Evidence forN. gonorrhoeae-dependent increasedC. muridaruminfectivity has thus not been demonstrated in murine coinfection, and the ability ofC. muridarumcoinfection to potentiateN. gonorrhoeaeinfectivity may depend on actively replicating vaginalC. muridarum. The proportion of mice with increased vaginal neutrophils (PMNs) was higher inN. gonorrhoeae-infected than inC. muridarum-infected mice, as expected, while that ofC. muridarum/N. gonorrhoeae-coinfected mice was intermediate to the singly infected groups, suggesting latentC. muridarummurine infection may limit PMN response to subsequentN. gonorrhoeaeinfection.IMPORTANCEOur work builds upon the limited understanding ofC. muridarum/N. gonorrhoeaecoinfection. Previously,N. gonorrhoeaeinfection of mice with acute (actively replicating) vaginalC. muridaruminfection was shown to increase recovery of viable vaginalN. gonorrhoeaeand vaginal PMNs, with no effect onC. muridarumvaginal shedding (R. A. Vonck et al., Infect Immun 79:1566–1577, 2011). It has also been shown that chlamydial infection of human and murine PMNs prevents normal PMN responses, including the response toN. gonorrhoeae(K. Rajeeve et al., Nat Microbiol 3:824–835, 2018). Our findings show no effect of latent genitalC. muridaruminfection on the recovery of viableN. gonorrhoeae, in contrast to the previously reported effect of acuteC. muridaruminfection, and suggesting that acute versus latentC. muridaruminfection may have distinct effects on PMN function in mice. Together, these studies to date provide evidence thatChlamydia/N. gonorrhoeaesynergistic interactions may depend on the presence of replicatingChlamydiain the genital tract, while chlamydial effects on vaginal PMNs may extend beyond acute infection.