Mouse prostacyclin (mIP) receptors transiently expressed in Chinese hamster ovary (CHO) cells activated both adenylyl cyclase and phospholipase C, with a 33-fold preference for signaling through Gs. The prostacyclin (IP) receptor agonists cicaprost, iloprost, carbacyclin, and prostaglandin E1 showed a similar order of potency for activation of both signaling pathways in cells transiently transfected with the mIP and the chimeric prostacyclin/prostaglandin D2 (IPN-VII/DPC and IPN-V/DPVI-C) receptors. Substitution of the carboxyl-terminal tail of the prostacyclin receptor with the corresponding region of the mDP receptor (IPN-VII/DPC) produced a receptor with increased coupling to both Gs and Gq. However, this increased G-protein coupling was lost in the IPN-V/DPVI-C receptor. The observation that both these chimeric receptors can activate phospholipase C indicates that the carboxyl-terminal tail of the IP receptor is not entirely responsible for its ability to couple to Gq. Site-directed mutagenesis studies suggest that isoleucine at position 323 in the IPN-VII/DPC receptor plays an important role in mediating the increased potency of this chimeric receptor.