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Background In humans, measurement of serum thyroid-stimulating hormone ( TSH) concentration is commonly used as a first-line discriminatory test of thyroid function. Recent reports indicate that canine TSH ( cTSH) assays can be used to measure feline TSH and results can help diagnose or exclude hyperthyroidism. Objectives To investigate the usefulness of cTSH measurements as a diagnostic test for cats with hyperthyroidism. Animals Nine hundred and seventeen cats with untreated hyperthyroidism, 32 euthyroid cats suspected of having hyperthyroidism, and 131 clinically normal cats. Methods Prospective study. Cats referred to the Animal Endocrine Clinic for suspected hyperthyroidism were evaluated with serum T4, T3, free T4 ( fT4), and TSH concentrations. Thyroid scintigraphy was used as the gold standard to confirm or exclude hyperthyroidism. Results Median serum TSH concentration in the hyperthyroid cats (<0.03 ng/ mL) was significantly ( P < .001) lower than concentrations in clinically normal cats (0.05 ng/ mL) or euthyroid cats with suspected thyroid disease (0.06 ng/ mL). Only 18 (2.0%) hyperthyroid cats had measurable TSH concentrations (≥0.03 ng/ mL), whereas 114 (69.9%) of the 163 euthyroid cats had detectable concentrations. Combining serum TSH with T4 or fT4 concentrations lowered the test sensitivity of TSH from 98.0 to 97.0%, but markedly increased overall test specificity (from 69.9 to 98.8%). Conclusions and Clinical Importance Serum TSH concentrations are suppressed in 98% of hyperthyroid cats, but concentrations are measurable in a few cats with mild-to-moderate hyperthyroidism. Measurement of serum TSH represents a highly sensitive but poorly specific test for diagnosis of hyperthyroidism and is best measured in combination with T4 and fT4. [ABSTRACT FROM AUTHOR]