Canine thyroid-stimulating hormone (cTSH), total thyroxine (T4) and free T4 by equilibrium dialysis (fT4d) were measured in serum samples from 107 dogs with clinical signs suggestive of hypothyroidism in which the diagnosis was either confirmed (n=30) or excluded (n=77) by exogenous TSH response testing. Median serum total T4 and fT4d concentrations were significantly lower and cTSH significantly higher (P<0·001) in hypothyroid compared with euthyroid dogs. Differential positive rate analysis determined optimal cut-off values of less than 14·9 nmol/litre (total T4), less than 5·42 pmol/litre (fT4d), greater than 0·68 ng/ml (cTSH), less than 17·3 (T4 to cTSH ratio), and less than 7·5 (fT4d to cTSH ratio) for hypothyroidism. These had a sensitivity and specificity of 100 and 75·3 per cent, 80 and 93·5 per cent, 86·7 and 81·8 per cent, 86·7 and 92·2 per cent, and 80 and 97·4 per cent, respectively, for diagnosing hypothyroidism. Corresponding areas under the receiver operating characteristic curves were 0·92, 0·93, 0·87, 0·93 and 0·93. Unexpectedly low cTSH values in hypothyroid dogs may have resulted from concurrent non-thyroidal illness. Unexpectedly high serum cTSH values in the euthyroid dogs might have resulted from recovery from illness or concurrent potentiated sulphonamide therapy. Measurement of endogenous cTSH concentration is a valuable diagnostic tool for canine hypothyroidism if used in association with assessment of T4. Estimation of fT4d added only limited additional information over total T4 measurement.