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Estradiol concentrations are evaluated in canine serum as part of an adrenal panel used to diagnose atypical Cushing's syndrome and other endocrine abnormalities. Estradiol concentrations are often elevated in dogs without clinical signs of hyperestrogenism, and the significance of this elevation is unknown. The purpose of this study was to estimate the variation in estradiol concentrations in normal dogs. Ten neutered male and female dogs were enrolled in the study. Blood was collected from each dog at 2 h intervals, four times during a given day. This was repeated approximately 1 (week 2) and 5 weeks later (week 6). There was no attempt for a given dog to be started at the exact time or day each week. Results showed that estradiol concentrations ranged from 44.6 to 120.3 pg/mL with a mean of 70.4 pg/mL, which is greater than the upper limit of normal for our laboratory (69 pg/mL). The mean difference between the highest and lowest concentrations for each dog was 28.8 pg/mL, with a range of 12.5-53.5 pg/mL. Mean estradiol concentrations from week 6 (63.2 pg/mL) were significantly lower than those from week 1 (71.4 pg/mL; P=0.015) and week 2 (76.5 pg/mL; P=0.0004). These data show a wide range of variability in estradiol concentration both within and between dogs and that these measurements often exceed the normal ranges established by the laboratory. Therefore, diagnosis of hyperestrogenism or atypical Cushing's syndrome based on increased estradiol concentrations should require compatible clinical presentation of hyperestrogenism together with elevated serum estradiol.