The majority of constitutively activating human TSH receptor (hTSHR) mutations are located in the transmembrane helices as well as in the extracellular (ECLs) and intracellular loops. S(281) is one of two positions in the ectodomain in which activating hTSHR mutations have been identified in vivo (S(281)T, I, and N). To investigate the functional properties of this key residue in more detail, S(281) was replaced by each of the other 19 amino acids. Many substitutions led to constitutive receptor activation, suggesting that S(281) plays a pivotal role in maintaining the receptor in its inactive state. Strikingly, all substitutions with aromatic residues (S(281)W, F, Y, and H) show expression similar to that of wild-type hTSHR and are tolerated at this position because they maintain basal activity or express only slight constitutive activity. Three-dimensional modeling of the hTSHR suggested that S(281) and surrounding residues are in close proximity to ECL1. To investigate the possible importance of an aromatic environment between the ectodomain in the vicinity of S(281) and ECL1, aromatic residues Y(279), Y(476), H(478), Y(481), Y(482), and H(484) were replaced by alanine. Functional characterization showed impaired cell surface expression and signaling for Y(279)A and Y(481)A, in contrast to the other alanine mutants. However, substitutions of Y(279) and Y(481) with other aromatic residues exhibited surface expression and signaling comparable to wild-type hTSHR. Our results suggest that Y(279) in the extracellular domain and probably Y(481) in the ECL1 also are involved in an aromatic environment around S(281) in the hTSHR, which is important for functional receptor conformation and intramolecular receptor signaling.