OBJECTIVE To evaluate use of an assay for measuring serum concentration of canine thyroid-stimulating hormone (cTSH) as an aid for diagnosing thyroid disease in a population of dogs suspected of having hypothyroidism. DESIGN Case-cohort study. ANIMALS 62 healthy dogs and 49 dogs with clinical signs consistent with hypothyroidism (16 were hypothyroid and 33 were euthyroid with concurrent disease). PROCEDURE Samples from healthy dogs were used to establish a reference range for serum cTSH concentration. The 49 dogs were categorized as hypothyroid or euthyroid with concurrent disease on the basis of clinical signs, results of additional diagnostic and thyroid-stimulating hormone (TSH) response tests, and response to administration of levothyroxine sodium. Function of the thyroid gland was considered normal when serum total thyroxine (T4) concentration 6 hours after TSH administration was > 2.5 micrograms/dl. Hypothyroidism was diagnosed when serum T4 concentration after TSH administration was < or = 1.5 microgram/dl. RESULTS Serum cTSH concentration differed significantly among all 3 groups. Four of 33 (12%) euthyroid dogs had cTSH concentrations that were greater than the reference range, whereas 6 of 16 (38%) hypothyroid dogs had cTSH concentrations within the reference range. Specificity for serum cTSH concentration was 0.88 and sensitivity was 0.63. When interpreted in combination with serum T4 concentration, specificity increased to 1.0. CLINICAL IMPLICATIONS cTSH assay had good specificity for use in the diagnosis of hypothyroidism in dogs. Because this assay had low sensitivity, a diagnosis of hypothyroidism could not be excluded on the basis of a serum cTSH concentration that was within the reference range.