Alveolar type II cells increase lipogenesis and convert glycogen into the phospholipids of surfactant in the late term fetal lung. Recent studies suggest that CCAAT/enhancing-binding protein (C/EBP) isoforms and sterol regulatory element binding protein (SREBP)-1c regulate fatty acid synthesis in adult type II cells in vitro. To define the temporal relationships and enzymes involved in lipogenesis in fetal rat lung, the mRNA levels of selected transcription factors and enzymes were determined. There was an increase in the mRNA levels of C/EBPalpha, C/EBPbeta, C/EBPdelta, peroxisomal proliferator-activated receptor gamma (PPARgamma), and SREBP-1c, but not SREBP-1a or SREBP-2 from fetal Days 19-21. There was also an increase in the mRNA levels of fatty acid synthase, stearoyl-CoA desaturase 1 (SCD-1), fatty acid translocase, glycerol-3-P acyl transferase, and phosphatidate cytidylyltransferase. By in situ hybridization, there was detectible expression of fatty acid synthase, SCD-1, and C/EBPalpha along the alveolar septae with the same distribution pattern as surfactant protein-C, whereas PPARgamma expression appeared to be restricted to macrophages. Regulation of lipogenesis at the mRNA level is predominately on enzymes of fatty acid synthesis and appears to be regulated by C/EBPalpha and SREBP-1c. SCD-1 and phosphatidate cytidylyltransferase are important components of the lipogenic response in the fetal lung that have not been recognized previously.