BACKGROUND: Measurement of salivary cortisol is a useful diagnostic test for hypercortisolism (HC) in humans. OBJECTIVES: To determine whether measurement of salivary cortisol concentration is a practical alternative to plasma cortisol to diagnose HC, to validate the use of salivary cortisol, and to examine the effect of time of day and sampling location on salivary cortisol. ANIMALS: Thirty healthy dogs and 6 dogs with HC. METHODS: Prospective, observational clinical trial including healthy volunteer dogs and dogs newly diagnosed with HC. Salivary and plasma cortisol concentrations were measured with an immunoassay analyzer. Intra- and interassay variability, linearity, and correlation between salivary and plasma cortisol concentrations were determined. RESULTS: The required 300 microL of saliva could not be obtained in 88/326 samples from healthy dogs and in 15/30 samples from dogs with HC. The intra-assay variability for measurement of salivary cortisol was 5-17.7%, the interassay variability 8.5 and 17.3%, and the observed to expected ratio 89-125%. The correlation (r) between salivary and plasma cortisol was 0.98. The time of day and location of collection did not affect salivary cortisol concentrations. Dogs with HC had significantly higher salivary cortisol values than healthy dogs (10.2 +/- 7.3 nmol/L versus 1.54 +/- 0.97 nmol/L; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: The ROCHE Elecsys immunoassay analyzer correctly measured salivary cortisol in dogs. However, a broad clinical application of the method seems limited, because of the large sample volume required.