We aimed to test the hypothesis that human umbilical cord blood (CB)-derived Stem/Progenitor cells (S/P) may participate in post myocardial infarction (MI) healing and regeneration. S/P cells were isolated from CB of male newborns by the Miltenye Biotec’s magnetic cell separation device (MACS) and stored for transplantation or expanded in the presence of a copper chelator and four early hematopoietic acting cytokines (SCF, TPO, FLt-3 and IL6). Under these culture conditions the proportion of stem (CD34+Lin-) and early progenitor (CD34+CD38-) cells increased by 73.3 and 251.7 fold, respectively (n=9). An MI model was established in athymic nude rats by permanent ligation of the left anterior descending coronary artery. Fresh or ex vivo expanded CB S/P cells (1-2x106 cells/rat) or saline (control) were injected I.V (CD133+ progenitors) or at the scar tissue (CD34+) 24 hours or one week respectively, post MI. By 4 weeks after cell therapy, the hearts were harvested and representative sections were either fixed or frozen sectioned. The presence of human-donor cells in recipient hearts was confirmed by fluorescent in situ hybridization (FISH) using deoxyribonucleic acid probes specific for human X and Y chromosomes or immunostaining for HLA-DR. HLA immunostaining revealed that both the fresh and the ex vivo expanded human CB derived donor cells engrafted and colonized the infracted myocardium. Positive HLA staining and Y signals were observed in vessel walls and cardiomyocytes, suggesting differentiation or fusion of the human CB donor cells into vasculature or myogenic cells. Examination of representative slides from control hearts was negative for both HLA and human sex chromosomes. Blinded computerized image analysis of representative slides stained for smooth-muscle alpha-actin detected a higher density (vessels/mm2) of capillaries and arterioles in the directly injected vs. control hearts (125 ± 15 vs. 44 ± 15/ mm2, p=0.02). Serial echocardiographic studies before and 4 weeks post cell therapy revealed that only directly injected ex vivo expanded CB cells were able to prevent LV dilatation accompanied by progressive LV dysfunction. In conclusion, our data suggest that S/P cells derived from human umbilical cord blood can engraft and colonize the infracted myocardium. Local delivery of ex vivo expanded CB derived cells, but not systemic injection, enhance new vessel formation and prevention of LV dilatation and myocardial dysfunction.