The role of host defenses in the pathogenesis of pelvic inflammatory disease (PID) remains largely uncharacterized. The antimicrobial peptides defensins are important components of innate host defense. To explore the relationship between neutrophil defensins and upper genital tract infection, 377 women who were at risk for PID were enrolled in a study. Women infected with Neisseria gonorrhoeae, Trichomonas vaginalis, or Chlamydia trachomatis had higher median levels of neutrophil defensins (human neutrophil peptides 1-3) in the vagina than did uninfected women. Neutrophil defensins were strongly associated with the presence of endometritis after the analysis was controlled for the presence of sexually transmitted diseases. Vaginal neutrophils were associated with endometritis only in the presence of elevated defensin levels, which highlights the importance of neutrophil activation, rather than the presence of neutrophils alone, in this inflammatory process. Neutrophil defensins appear to participate in the host defense in ascending pelvic infection and the pathogenesis of PID.