The present study was performed to determine the accuracy and reproducibility of calculating the mitral regurgitant orifice area with the proximal isovelocity surface area (PISA) method in dogs with experimental mitral regurgitation and in canine patients with chronic mitral insufficiency and to evaluate the effect of general anesthesia on mitral regurgitation. Eight adult, Beagle dogs for experimental mitral regurgitation and 11 small breed dogs with spontaneous mitral regurgitation were used. In 8 Beagle dogs, mild mitral regurgitation was created by disrupting mitral chordae or leaflets. Effective regurgitant orifice (ERO) area was measured by the PISA method and compared with the measurements simultaneously obtained by quantitative Doppler echocardiography 4 weeks after creation of mitral regurgitation. The same procedure was performed in 11 patients with isolated mitral regurgitation and in 8 Beagle dogs under two different protocols of general anesthesia. ERO and regurgitant stroke volume (RSV) by the PISA method correlated well with values by the quantitative Doppler technique with a small error in experimental dogs (r = 0.914 and r = 0.839) and 11 patients (r = 0.990 and r = 0.996). The isoflurane anesthetic echocardiography demonstrated a significant decrease of RSV, and there was no significant change in fractional shortening (FS), ERO area, LV end-diastolic and LV end-systolic volume. ERO area showed increasing tendency after ketamine-xylazine administration, but not statistically significant. RSV, LV end-systolic and LV end-diastolic volume increased significantly (p < 0.01), whereas FS significantly decreased (p < 0.01). The PISA method is accurate and reproducible in experimental mitral regurgitation model and in a clinical setting. ERO area is considered and preferred as a hemodynamic-nondependent factor than other traditional measurements.