Abstract Fetal fibroblast cell culture from cotyledons of bovine placenta and animal experiments close to term were used to elucidate afterbirth release and factors missing in the signal transduction mechanism for retained fetal membranes (RFM) after delivery. In cell culture the addition of arachidonic acid (Ara) to the medium caused rapid release to free floating cell in the culture dish, accompanied by matrix metalloproteinase (MMP) activation, being consistent with previous in vivo observations, where a relation between MMP and fetal membrane release had been shown. Ara-induced cell floating was not inhibited by the addition of cyclooxygenase (COX) inhibitor, and not induced by the addition of PGF2α or PGE2 to replace Ara, while 12-lipoxygenase (12-LOX) metabolite of Ara, 12-oxo-eicosatetraenoic acid (12-oxoETE), strongly induced cell floating. In the animal experiments, 12-oxoETE injection to delivery-induced cows ( n  = 6) using prostaglandin (PG) and dexamethazone resulted in rapid release of fetal membranes. In cows with natural calf delivery, a 12-oxoETE peak (11.7–16.8 ng/ml) was observed in maternal blood plasma prior to release of fetal membranes. This investigation thus gives new indications for that the mediator for fetal membrane release is 12-oxoETE and not PG.