Relaxation of vascular smooth muscle and prevention of blood coagulation are mediated by ligand-induced activation of the human prostacyclin (hIP) receptor, a seven-transmembrane-domain G-protein-coupled receptor (GPCR). In this study, we elucidate the molecular requirements for receptor activation within the region of the ligand-binding pocket, identifying transmembrane residues affecting potency. Eleven of 30 mutated residues in the region of the ligand-binding domain exhibited defective activation (decreased potency). These critical residues localized to four distinct clusters (analysis via a rhodopsin-based human prostacyclin receptor homology model). Residues Y75 2.65 (TMII), F95 3.28 (TMIII), and R279 7.40 (TMVII) comprised the immediate binding-pocket cluster and were shown to be essential for proper receptor activation, compared to equivalent expression levels of the wild-type hIP (WT EC 50 = 1.2 ± 0.1 nM; Y75 2.65 A EC 50 = 347.3 ± 62.8 nM, p < 0.001; F95 3.28 A EC 50 = 8.0 ± 0.6 nM, p < 0.001; R279 7.40 A EC 50 = 130 ± 63.0 nM, p < 0.001). Residues S20 1.39 (TMI), F24 1.43 (TMI), and F72 2.62 (TMII) were localized to a cluster involving P17 1.36, a critical residue thought to facilitate transmembrane movement during changes in activation conformation. A third cluster formed around amino acid D60 2.50 (TMII), containing the highly conserved (100% of prostanoid receptors) D288 7.49/P289 7.50 motif located in TMVII. Last, a large hydrophobic cluster composed of aromatic residues F146 4.52 (TMIV), F150 4.56 (TMIV), F184 5.40 (TMV), and Y188 5.44 (TMV) was observed away from the ligand-binding pocket, but still necessary for hIP activation. These results assist in delineating the potential molecular requirements for agonist-induced signaling through the transmembrane domain. Such observations may be generally applicable, as many of these clusters are highly conserved among the prostanoid receptors as well as other class A GPCRs.