We previously reported that administration of dexamethasone to the pregnant dam increased the activity of fatty-acid synthase (EC 188.8.131.52) in fetal rat lung and that this effect was reduced when triiodothyronine (T3) was also administered. To determine whether the hormones act directly on the lung, we examined their effects in organ culture. Explants of 18-day and 19-day fetal rat lung were cultured with 100 nM dexamethasone or 100 nM T3, the two hormones together or no hormone at all for 48 h, after which fatty-acid synthase was assayed. Dexamethasone increased fatty-acid synthase activity at both gestational ages. T3 alone had no effect on 18-day, but decreased the activity in 19-day explants by 20%. T3 reduced the stimulatory effect of dexamethasone from 177% to 102% and from 61% to 22% in 18- and 19-day explants, respectively. The effects of dexamethasone and T3 were concentration dependent, with EC50 (concentration achieving 50% of the maximum effect) values of 0.65 nM and approx. 25 nM, respectively. This dexamethasone EC50 is lower than the reported Kd for dexamethasone binding, but the T3 EC50 is considerably higher than its reported Kd. The physiological significance of the T3 effect is, therefore, not clear. The effect of dexamethasone was not apparent until at least 12 h after exposure to the hormone and it was abolished by actinomycin D. Immunoprecipitation with antibody against rat liver fatty-acid synthase showed that there was more fatty-acid synthase in the dexamethasone-treated than in the control cultures. The potency order of glucocorticoids in stimulating fatty-acid synthase was similar to that previously reported for specific nuclear glucocorticoid binding. These data show that dexamethasone and T3 act directly on the fetal lung and that the stimulatory effect of the glucocorticoid on fatty-acid synthase is due to new protein synthesis.