The contribution of acetoacetate (AcAc), beta-hydroxybutyrate (beta OHB), lactate and glucose to pulmonary surfactant lipid synthesis in three- to five-day-old rats was measured. Minced lung tissue was incubated with 3H2O and [3-14C]AcAc, [3-14C]beta OHB, [U-14C]lactate or [U-14C]glucose, and the radioactivity incorporated into surfactant lipids was measured. When expressed as nmol of substrate incorporated/g lung tissue per four hr, lactate was incorporated more rapidly than other substrates into total surfactant lipids and phosphatidylcholine (PC). There was no difference in the rates of incorporation of lactate, AcAc or glucose into disaturated PC (DSPC). Substrates other than glucose were incorporated almost exclusively into fatty acids, whereas 60-80% of glucose incorporated into surfactant phospholipids was found in fatty acids, with the remaining in glyceride-glycerol. When expressed as nmol acetyl units incorporated/g lung tissue per four hr, the rates of AcAc, lactate and glucose incorporation into total surfactant fatty acids were comparable. Glucose incorporation into DSPC and PC was greater than that of AcAc and lactate. When glucose was the only exogenous substrate added to the incubation medium, it contributed 37% of total surfactant fatty acids synthesized de novo. In the presence of other substrates, the contribution of glucose to de novo fatty acid synthesis dropped to 14-20%. In the presence of unlabeled glucose, 14C-labeled AcAc, lactate and beta OHB contributed 52%, 40% and 19%, respectively, of the total fatty acids synthesized de novo. The rate of beta OHB incorporation into surfactant lipids was only about 50% that of other substrates and was accompanied by low activity of beta-hydroxybutyrate dehydrogenase measured for newborn lung.(ABSTRACT TRUNCATED AT 250 WORDS)