Regulation of circulating thyroid hormones is a complex process that involves the hypothalamus, pituitary gland, thyroid gland, plasma transport proteins, and cellular uptake and metabolism of thyroid hormones. The hypothalamus produces thyrotropin-releasing hormone (TRH), which stimulates pituitary secretion of thyrotropin (TSH) and enhances its bioactivity. Thyroid hormone secretion is controlled primarily by TSH, which stimulates both synthesis and release of thyroxine (T4) and 3,5,3′-triiodothyronine (T3). Thyroid hormones in turn act in a negative feedback manner to decrease TSH and TRH secretion. Additional factors opposing secretion of TSH include somatostatin, dopamine, and other catecholamines, and cytokines such as tumor necrosis factor and some interleukins. After entering the circulation, thyroid hormones are highly protein bound, and only the unbound, or ‘‘free,’’ hormone enters cells by both passive diffusion and receptor-mediated transport. Once inside the cell, T3 binds to nuclear receptors and initiates the action of thyroid hormone on that cell. Thyroxine undergoes deiodination to T3 before binding to the thyroid hormone receptor and can also be deiodinated to form the metabolically inactive 3,3′,5′-triiodothyronine (reverse T3). Further deiodination of T3 results in less-active metabolites. Alternative routes of metabolism of thyroid hormones, including deamination, decarboxylation, sulfation, and glucuronidation, may account for substantial metabolism in some species, including the dog. Disturbances of any of the aforementioned factors that determine thyroid hormone secretion, transport, distribution, and metabolism can alter thyroid function tests.