G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA(2)) receptor (TP) and prostacyclin (PGI(2)) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease.