We investigated the biosynthesis of phospholipid, neutral lipids, glycoproteins, and DNA in primary cultures of rat oxyntic mucosal cells. In addition, responses of these biosynthetic pathways to the gastric protective agent 16,16-dimethyl prostaglandin E2 (dmPGE2) were studied. Cultured gastric cells under control conditions synthesized glycoprotein in a linear manner over time. The cells responded to dmPGE2 with an increase in glycoprotein synthesis without an effect on DNA synthesis. Investigations of lipid synthesis showed that phospholipid was produced in a linear fashion by these cells, however, no effect of exogenously administered dmPGE2 on its rate of formation was discernible. In contrast, the incorporation of labeled palmitate into neutral lipids revealed that triglyceride biosynthesis was significantly increased by the addition of dmPGE2 to the culture medium, which could be further enhanced by the administration of the phosphodiesterase inhibitor, isobutyl methyl xanthine. Cyclic nucleotide involvement was further suggested by our finding that triglyceride synthesis in cultured gastric mucous cells could be increased a comparable amount by the addition of both dbcAMP and dbcGMP to the medium. The possible relationship between these biochemical alterations and the gastric protective action of dmPGE2 is discussed.