We recently presented a new method for quantification of CArdiac FUnction - denoted CAFU - as the first step in the development of an automated method for integrated interpretation of gated myocardial perfusion single photon emission computed tomography (SPECT) images. The aim of this study was to validate CAFU in the assessment of global and regional function of the left ventricle. Quantitative gated-SPECT (QGS), the most widely used software package for quantification of gated-SPECT images, was used as reference method for the measurements of ejection fraction (EF) and ventricular volumes, and visual analysis by an experienced physician was used as reference method for the measurements of regional wall motion and thickening. Two different groups of consecutive patients referred for myocardial perfusion scintigraphy were studied. Global function was evaluated in 316 patients and regional function in 49 other patients. The studies were performed using a 2-day stress/rest 99 m-Tc-sestamibi protocol. A good correlation was found between EF values from QGS and CAFU (EF CAFU = 0.84 EF QGS + 13, r = 0.94), but CAFU values were on average 4 EF points higher than QGS values. With CAFU the segments with normal thickening according to the physician showed significantly higher thickening values (in all parts of the myocardium) compared to the segments classified as having abnormal thickening. In conclusion, this study demonstrates that CAFU can be used to quantify global and regional function in gated-SPECT images. This is an important step in our development of an automated method for integrated interpretation of gated-SPECT myocardial perfusion scintigraphy studies.