Abstract The aim of the present study was to investigate the effects of the lipid mediator, ceramide, on prostaglandin and cytokine production by human placental, choriodecidual, and amnion cells in culture, using the cell-permeable ceramide analog, N-acetyl-sphingosine (C 2-ceramide). Cells derived by enzymatic dispersion of choriodecidual, placental, and amnion tissues from placentas delivered by caesarian section at term were exposed to C 2-ceramide (1–25 μM) with or without TNF-α (10 ng/mL) in serum-free media over 16 h ( n = 4 wells/treatment). C 2-ceramide (25 μM) stimulated PGE 2 production in choriodecidual, placental, amnion, and amnion-derived WISH cell cultures to 198.7 ± 22.2%, 418.7 ± 42.9%, 595.5 ± 96.1%, and 517.8 ± 42.1% of control, respectively (mean ± SEM, from 3–7 experiments). C 2-ceramide similarly augmented prostaglandin production in TNF-α-stimulated cells. In contrast, production of IL-6 and IL-8 was not stimulated by C 2-ceramide. Time-course studies revealed that the effects of C 2-ceramide on PGE 2 production were much slower than those of TNF-α. The degree of stimulation of PGE 2 production elicited by C 2-ceramide was maintained over a wide range of TNF-α concentrations. The ceramide synthesis inhibitor fumonisin B 1 had only moderate inhibitory effects on TNF-α-stimulated PGE 2 production. Taken together, these studies suggest that although ceramide has the ability to stimulate PG production by gestational tissues, ceramide release is unlikely to play a major role in mediating the pro-inflammatory effects of TNF-α in these tissues.