In the present study we examined whether the vitamin D3 metabolites, 25-hydroxyvitamin D and 1-25-dihydroxycholecalciferol affected the production of the prostaglandins PGE2 and PGF2 alpha in human fetal membranes and placenta in vitro. Human amnion, chorion, decidual, and placental cells were maintained in primary monolayer culture. Treatment with the vitamin D3 metabolites resulted in an increase in PGE2 and PGF2 alpha production by amnion, decidua, and placental cells; however, these effects varied with time and were different between tissues. Although there was no significant increase in the production of PGE2 and PGF2 alpha by chorion cells in vitro, there was a significant increase in the production of prostaglandin F metabolites after treatment with the vitamin D3 metabolites. The data suggest that the vitamin D3 metabolites may increase free calcium availability and the conversion of arachidonic acid to the prostaglandins. The data do not, however, exclude the possibility that the vitamin D3 metabolites act at other points of arachidonic acid metabolism. These findings raise the possibility of a paracrine role for the vitamin D3 metabolites in the modulation of prostaglandin production within the human fetal membranes and placenta.