The effects of prostaglandins E1 and E2, indomethacin, arachidonic acid, and 8,11,14-eicosatrienoic acid on the rate of (14C)-acetate incorporation into lipids in mouse mammary gland explants were studied. Prostaglandins E1 and E2, as well as their precursors 8,11,14-eicosatrienoic acid and arachidonic acid, inhibited the rate of (14C)-acetate incorporation into lipids, possibly through increased cAMP levels or through end product inhibition of acetyl CoA carboxylase and fatty acid synthetase by long chain fatty acids. Indomethacin at concentrations of 50 micrograms/ml and above significantly reduced the basal rate of 14C-acetate incorporation into lipids, but it did not abolish the prolactin response. Since the prostaglandins, at low concentrations, have prolactin-like actions on RNA synthesis and ornithine decarboxylase activity, and since indomethacin attenuates the actions of prolactin on RNA synthesis, casein synthesis, and ornithine decarboxylase activity, it seems apparent that all of the metabolic actions of prolactin in the mammary gland may not occur via the same primary mechanism.