We have compared the metabolism of ( 3H) arachidonic acid by monolayers of human amnion, cells obtained prior to or following labor at term. Radiolabel was either added exogenously or previously incorporated into cellular phospholipid pools to compare metabolism of arachidonic acid from different substrate sources. Cells obtained both prior to and following labor synthesized metabolites co-chromatographing on HPLC with di- and mono-HETEs and also a metabolite with polarity corresponding to a epoxyeicosatrienoic acid. Both types of cells were able to synthesize PGE 2 when ( 3H) arachidonic acid was added exogenously. However, only those cells obtained following labor synthesized PGE 2 from ( 3H) arachidonic acid incorporated into intracellular pools. These findings suggest that the cyclooxygenase and PGE 2 isomerase enzymes are present in amnion prior to delivery but that exogenous arachidonic acid would be required for PGE 2 synthesis at that time as the enzymes do not appear to be linked to a source of endogenous arachidonic acid. At the time of parturition, there may be a switching on of an enzyme system to generate arachidonic acid from intracellular pools specifically for PGE 2 synthesis or alternatively coupling of such a system to a cyclooxygenase-PGE 2 isomerase system resulting in PGE 2 synthesis. These findings raise intriguing new possibilities for the regulation of eicosanoid synthesis in amnion which may include membrane topography, substrate pool-enzyme linking and regulation of specific phospholipase enzymes.