BACKGROUND: Intra-amniotic lipopolysaccharide (LPS) exposure may affect neonatal outcome by altering fetal lung and immune system development. We hypothesized that intra-amniotic LPS exposure would cause persistent fetal pulmonary responses as the lungs develop in utero. METHODS: Fetal lambs were exposed to intra-amniotic LPS at 118 or at 118 and 123 d of gestational age (GA) with delivery at 125, 133, or 140 d (term = 147 d). Immune responses, PU.1 expression, Toll-like receptor (TLR)-1,2,4,6 mRNA levels, mast cell levels, and pulmonary elastin deposition were evaluated. RESULTS: After a single dose of LPS, pulmonary inflammatory responses were observed with increases of (i) PU.1 and TLR1 at 125 d GA and (ii) monocytes, lymphocytes, TLR2, and TLR6 at 133 d GA. Repetitive LPS exposure resulted in (i) increases of neutrophils, monocytes, PU.1, and TLR1 at 125 d GA; (ii) increases of neutrophils, PU.1, and TLR2 at 133 d GA; and (Hi) decreases of mast cells, elastin foci, TLR4, and TLR6 at early gestation. At 140 d GA, only PU.1 was increased after repetitive LPS exposure. CONCLUSION: The preterm fetal lung can respond to a single exposure or repeated exposures from intra-amniotic LPS in multiple ways, but the absence of inflammatory and structural changes in LPS-exposed fetuses delivered near term suggest that the fetus can resolve an inflammatory stimulus in utero with time.