Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased (∼4-fold) with term labor in amnion (n = 15), and with preterm labor (∼30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r² = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1β and tumor necrosis factor α. Production of ENA-78 by choriodecidual explants was increased modestly after 2–4 h of exposure to lipopolysaccharide (5 μg/ml). An immunoreactive doublet (∼8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.