BACKGROUND: The identification and isolation of human cardiac progenitor cells (hCPCs) offer new approaches for myocardial regeneration and repair. Still, the optimal source of human cardiac progenitor cells and the influence of patient characteristics on their number remain unclear. Using a novel method to isolate human cardiac progenitor cells, we aimed to define the optimal source and association between their number and patient characteristics. METHODS AND RESULTS: We developed a novel isolation method that produced viable cells (7 x 10(6)+/-6.53 x 10(5)/g) from various tissue samples obtained during heart surgery or endomyocardial biopsies (113 samples from 94 patients 23 to 80 years of age). The isolated cardiac cells were grown in culture with a stem cell expansion medium. According to fluorescence-activated cell sorting analysis, cultured cells derived from the right atrium generated higher amounts of c-kit(+) (24+/-2.5%) and Islet-1(+) cells (7%) in culture (mean of passages 1, 2, and 3) than did cultured cells from the left atrium (7.3+/-3.5%), right ventricle (4.1+/-1.6%), and left ventricle (9.7+/-3%; P=0.001). According to multivariable analysis, the right atrium as the cell source and female sex were associated with a higher number of c-kit(+) cells. There was no overlap between c-kit(+) and Islet-1 expression. In vitro assays of differentiation into osteoblasts, adipocytes, and myogenic lineage showed that the isolated human cardiac progenitor cells were multipotent. Finally, the cells were transplanted into infarcted myocardium of rats and generated myocardial grafts. CONCLUSIONS: Our results show that the right atrium is the best source for c-kit(+) and Islet-1 progenitors, with higher percentages of c-kit(+) cells being produced by women.