The human platelet thromboxane A2 receptor is a member of the G-protein- coupled superfamily of receptors. Previous pharmacologic studies examining the effects of biochemical reduction, oxidation, or sulfhydryl alkylation on thromboxane receptors have suggested a role for cysteines in determining receptor binding characteristics. To characterize the roles of individual cysteines, we employed site-directed mutagenesis to substitute serines for cysteines at seven positions throughout the human K562 thromboxane receptor and analyzed mutant receptor radioligand ([1S-(1α,2β(5Z),3α(1E,3S),4α]- 7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2-2-1]heptane-2- yl]-5-heptenoic acid) binding and calcium signaling. Replacing cysteines in the amine terminus (amine acid position 11), and transmembrane domains two and six (positions 60 and 257) had little effect on thromboxane receptor binding or signaling. Introduction of serines for cysteines in the first (position 105) or the second (position 183) extracellular loop eliminated thromboxane receptor binding, consistent with the existence of a critical disulfide bond between these positions. Mutation of a second cysteine in extracellular loop one (position 102) resulted in a receptor with decreased binding affinity and low binding capacity that transduced only a low amplitude calcium signal, suggesting the involvement of a free sulfhydryl group at this location in receptor-ligand interactions. Finally, mutation of the cysteine at position 223, located in intracellular loop three, resulted in a receptor with normal ligand binding characteristics, but which did not transduce a calcium signal. Some additional amine acid substitutions in this region of the receptor (Cys-223 → Ala, Thr-221 → Met) resulted in receptors that had normal binding but transduced low amplitude calcium signals, while other mutations in the same region (His-224 → Arg and His-227 → Arg) exhibited normal binding and calcium signaling characteristics. These findings demonstrate that cysteines in extracellular loops one and two contribute to proper ligand binding to thromboxane receptors and show the importance of discrete amine acid sequences in the third intracellular loop, especially cysteine 223, in thromboxane receptor-effector coupling.