TSH and the interaction with its receptor (TSHR) in the thyroid gland play a crucial role in the pituitary-thyroid axis of all vertebrates. Released upon stimulation by TSH, thyroid hormones influence numerous processes in the body and are extremely important during the last week of chicken embryonic development. In this study, we have cloned and functionally characterized the chicken TSHR (cTSHR), which was found to be a G protein-coupled receptor consisting of 10 exons. Besides the full-length cDNA, we detected two splice variants lacking either exon 3, or exons 2 and 3, both part of the extracellular domain of the receptor. Bovine TSH increased intracellular cAMP levels in HEK-239 cells transiently expressing the full-length cTSHR (EC50 = 1.43 nm). In situ hybridization showed the expression of cTSHR mRNA in the thyroidal follicular cells. cTSHR mRNA expression, as determined by real-time PCR, was also found in several other tissues such as brain, pituitary, pineal gland, and retina, suggesting that the TSH-TSHR interaction is not only important in regulating thyroid function. TSHR mRNA expression in the thyroid gland did not change significantly during the last week of embryonic development, which suggests that an increased thyroidal sensitivity is not part of the cause of the concomitant increasing T4 levels.