Objective-To validate the use of a human enzyme immunoassay (EIA) kit for measurement of plasma antidiuretic hormone (ADH) concentration in dogs and evaluate plasma ADH concentrations in dogs with congestive heart failure (CHF) attributable to acquired cardiac disease, compared with findings in healthy dogs. Animals-6 healthy dogs and 12 dogs with CHF as a result of chronic degenerative valve disease or dilated cardiomyopathy. Procedures-Plasma samples from the 6 healthy dogs were pooled and used to validate the EIA kit for measurement of plasma ADH concentration in dogs by assessing intra-assay precision, dilutional linearity, and spiking recovery. Following validation, plasma ADH concentrations were measured in the 6 healthy dogs and in the 12 dogs with CHF for comparison. Results-The EIA kit measured ADH concentrations in canine plasma samples with acceptable intra-assay precision, dilutional linearity, and spiking recovery. The intra-assay coefficient of variation was 11%. By use of this assay, the median plasma concentration of ADH in dogs with CHF was 6.15 pg/mL (SD, 3.2 pg/mL; range, 4.18 to 15.47 pg/mL), which was significantly higher than the median concentration in healthy dogs (3.67 pg/mL [SD, 0.93 pg/mL; range, 3.49 to 5.45 pg/mL]). Conclusions and Clinical Relevance-Plasma ADH concentrations in dogs can be measured with the tested EIA kit. Plasma ADH concentrations were higher in dogs with CHF induced by acquired cardiac disease than in healthy dogs. This observation provides a basis for future studies evaluating circulating ADH concentrations in dogs with developing heart failure.