Previous studies have shown that triiodothyronine (T3) enhances the effect of dexamethasone on phosphatidylcholine (PC) synthesis in organ cultures of fetal rat lung. The aim of this study was to investigate whether similar interactions occurred in vivo and to explore possible mechanisms for this phenomenon. Injection of 7.0 mg/kg T3into pregnant rats on d 18 and 19 of gestation resulted in a mean fetal serum T3level of 2380 ng/dl on d 20 (control, 84 ng/dl) and in maximal (34%) stimulation of choline incorporation into PC. Injection of 1.0 mg/kg betamethasone using the same protocol as for T3resulted in maximal stimulation of 33% and administration of both hormones together produced a 69% increase, an additive affect. The percentage of PC that was disaturated was increased with betamethasone, but decreased with T3. Betamethasone treatment resulted in an increase in the whole lung disaturated PC content, but treatment with T3did not. Betamethasone administration also increased fetal serum T3levels, but T3injection did not produce elevated fetal serum corticosterone levels. Injection of T3in vivo, or exposure of explants of 18-d fetal lung to 100 nm T3for up to 48 h did not result in an increase in cytoplasmic glucocorticoid binding or nuclear translocation of the receptor steroid complex. Exposure of explants to glucocorticoid or T3in vivo or in culture (dexamethasone, 100 nM and T3, 100 nM; for 48 h) resulted in a significant increase in the activity of cholinephosphate cytidylyltransferase, an enzyme in the choline incorporation pathway of PC synthesis. Exposure of explants to the combination of hormones resulted in stimulation that was equal to the sum of that produced by the single hormones but was not statistically significantly different from the glucocorticoid effect. The activities of other enzymes of phospholipid synthesis were not increased by exposure to either hormone, in vivo or in vitro. The additive effects of T3and glucocorticoid with regard to choline incorporation into PC in fetal rat lung suggest that combined hormone therapy may be useful for the prevention of respiratory distress syndrome in humans. Further animal studies are required, however, before clinical use can be considered.