Ceramide has emerged as a novel second messenger for intracellular signalling. It is produced from sphingomyelin and is involved in the control of cell differntiation, proliferation, and apoptosis. C2- ceramide, short chain ceramide, plays a role in mediating contraction of cat esophageal smooth muscle cells. We examined the effect of synthesized ceramide analogues on the C2-ceramide and ACh-induced contraction in esophageal smooth muscle cells isolated with collagenase. CY3523, CY3525, or CY3723 inhibited C2-ceramide induced contraction, in a time dependent manne. Each analogue also inhibited the contraction in concentration dependent manners. CY 3523, CY 3525, and CY 3723 had no effect to the contraction induced by PMA. The inhibition with CY3523, CY3525 and CY3723 on the C2- ceramide induced contraction was recovered by PMA. These analogues decreased the density of MAPK bands (p44/42 or p38) in the western blot. These results suggest that ceramide analogues can inhibit C2-ceramide induced contraction via PKC and MAPK dependent pathway.