The authors collected urine specimens in 31 normal dogs, 25 dogs with hyperadrenocorticism, 21 dogs in which hyperadrenocorticism was suspected but was not present, and 28 dogs with a variety of severe, nonadrenal diseases. Cortisol and creatinine were measured in unextracted urine by radioimmunoassay and spectrophotometry, respectively, and the cortisol:creatinine ratio was calculated for each specimen. The mean ± SD urine cortisol:creatinine concentration ratio in the dogs with hyperadrenocorticism (103.1 ± 100.7) was significantly (P < 0.001) higher than that in the normal dogs (13.1 ± 7.0). The mean urine cortisohcreatinine ratio in dogs initially suspected of having hyperadrenocorticism (16.3 ± 7.0) was significantly (P < 0.001) lower than the ratio in dogs with hyperadrenocorticism, but was not significantly different than that in the normal dogs. The mean urinary cortisohcreatinine ratio in the dogs with nonadrenal disease (82.8 ± 97.7) was significantly (P < 0.001) higher than that in both the normal dogs and dogs in which hyperadrenocorticism was initially suspected, but was not different than the ratio in the dogs with hyperadrenocorticism. The sensitivity of the urine cortisohcreatinine ratio as a diagnostic test for hyperadrenocorticism was 0.92. The specificity was high in the normal dogs (0.97) and the dogs initially suspected of having hyperadrenocorticism (0.95), with ≤ 5% having false-positive results. However, the specificity was very low (0.21) in the dogs with moderate to severe nonadrenal disease, with 79% having false-positive results. Similarly, both positive and negative predictive values and diagnostic efficiency were high in the normal dogs and dogs suspected of having hyperadrenocorticism but were low in the dogs with nonadrenal illness. When the results of the cortisol assay used in the study were compared to results obtained by two other commercially available cortisol radioimmunoassays, a high correlation between results was found. The urine cortisohcreatinine ratio is a sensitive screening test for the detection of hyperadrenocorticism in dogs. As with other pituitary-adrenal function tests, however, the urine cortisohcreatinine ratio cannot be used to diagnose hyperadrenocorticism in dogs that have moderate to severe nonadrenal disease.