Background <p>Prematurity remains the leading cause of neonatal mortality and morbidity, largely due to pulmonary surfactant deficiency. We compared isolated and combined transamniotic delivery of surfactant proteins B (SPB) and C (SPC) mRNA.</p> Methods <p>Fetuses (<i>n</i> = 415) from thirty-two time-dated dams underwent intra-amniotic injections on gestational day 17 (E17, term= E21-22) of lipopolyplex-encapsulated mRNA consisting of either human SPB (hSPB) mRNA (<i>n</i> = 151), human SPC (hSPC) mRNA (<i>n</i> = 102), a combination of hSPB+hSPC mRNA (<i>n</i> = 113), or lipopolyplex without mRNA (<i>n</i> = 49). Fetal lungs were screened for hSPB and hSPC by ELISA daily until term. Phosphatidylcholine was measured in the amniotic fluid.</p> Results <p>Controlled by mRNA-free injections, the hSPB protein was detected in the SPB mRNA group only at E18 and E19, whereas in the SPB + SPC mRNA group it was detected at every preterm timepoint (E18-E20), either separately or combined (<i>p</i> &lt; 0.001 for all). The hSPC protein was detected in the SPC mRNA group only at E18 (<i>p </i>= 0.015). Amniotic fluid phosphatidylcholine was significantly increased in the SPC mRNA and SPB + SPC mRNA groups compared to controls (<i>p</i> &lt; 0.001).</p> Conclusions <p>Combined transamniotic delivery of exogenous mRNA encoding for SPB and SPC enhances surfactant production prior to term when compared with isolated mRNA administrations in a healthy rat model.</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>Transamniotic mRNA delivery may become a novel strategy for surfactant replacement therapy.</p> </ItemContent> <ItemContent> <p>Combined transamniotic mRNA delivery for surfactant proteins B and C enhances preterm surfactant production.</p> </ItemContent> <ItemContent> <p>Transamniotic mRNA delivery can be achieved in a short time frame, for example compatible with select clinical settings of preterm labor.</p> </ItemContent> </UnorderedList></p>