Objective - To determine the within-day and between-day variability of regurgitant fraction (RF) assessed by use of the proximal isovelocity surface area (PISA) method in awake dogs with degenerative mitral valve disease (MVD), measure RF in dogs with MVD, and assess the correlation between RF and several clinical and Doppler echocardiographic variables. Design - Prospective study. Animals - 6 MVD-affected dogs with no clinical signs and 67 dogs with MVD of differing severity (International Small Animal Cardiac Health Council [ISACHC] classification). Procedures - The 6 dogs were used to determine the repeatability and reproducibility of the PISA method, and RF was then assessed in 67 dogs of various ISACHC classes. Mitral valve regurgitation was also assessed from the maximum area of regurgitant jet signal-to-left atrium area (ARJ/LAA) ratio determined via color Doppler echocardiographic mapping. Results - Within- and between-day coefficients of variation of RF were 8% and 11%, respectively. Regurgitation fraction was significantly correlated with ISACHC classification and heart murmur grade and was higher in ISACHC class III dogs (mean+or-SD, 72.8+or-9.5%) than class II (57.9+or-20.1%) or I (40.7+or-19.2%) dogs. Regurgitation fraction and left atriumto-aorta ratio, fractional shortening, systolic pulmonary arterial pressure, and ARJ/LAA ratio were significantly correlated. Conclusions and Clinical Relevance - Results suggested that RF is a repeatable and reproducible variable for noninvasive quantitative evaluation of mitral valve regurgitation in awake dogs. Regurgitation fraction also correlated well with disease severity. It appears that this Doppler echocardiographic index may be useful in longitudinal studies of MVD in dogs.