Human neutrophil peptides 1–3 (defensins) are granule constituents released from activated neutrophils. We hypothesized that amniotic fluid (AF) defensin levels are elevated in preterm labor (PTL) patients with subclinical intrauterine infection (IUI). AF samples were obtained from 203 pregnant patients with varying clinical characteristics. Defensin levels were measured by enzyme-linked immunosorbent assay. Median AF defensin levels were fourfold to 24-fold higher in patients with IUI than in preterm and term controls. Among patients with subclinical IUI, the degree of AF defensin elevation was greater in those with a positive AF culture. AF defensin levels increased exponentially with increasing severity of histologic chorioamnionitis. An AF defensin level of >2,500 ng/mL identified 88% of patients with a positive AF culture, whereas a level of >400 ng/mL identified 85% of all infected patients. AF defensin levels accurately identify patients with subclinical IUI, as defined by a positive AF culture or placental histology.