We are investigating the effects of human umbilical cord blood mononuclear progenitor cells (HUCBC) for the treatment of acute myocardial infarction because human cord blood is a readily available and an abundant source of primitive cells that may be beneficial in myocardial repair. However, there is currently no scientific consensus on precisely when to inject stem/progenitor cells for the optimal treatment of acute myocardial infarction. We used an in vitro assay to determine the attraction of infarcted rat myocardium at 1, 2, 2.5, 3, 6, 12, 24, 48, and 96 h after left anterior descending coronary artery (LAD) occlusion from 45 rats for HUCBC in order to determine the optimal time to transplant HUCBC after myocardial infarction. Our assay is based on the migration of fluorescent DAPI-labeled HUCBC from wells in an upper chamber of a modified Boyden apparatus through a semiporous polycarbonate membrane into wells in a lower chamber that contain either normal or infarcted myocardium. DAPI-labeled HUCBC (100,000) were placed in each of the separate wells above the membrane that corresponded to normal or infarct homogenate in the lower wells. The greatest HUCBC migration to infarcted myocardium occurred at 2 h and 24 h after LAD occlusion in comparison with normal controls. A total of 76,331 +/- 3384 HUCBC migrated to infarcted myocardium at 2 h and 69,911 +/- 2732 at 24 h after LAD occlusion (both p < 0.001) and significantly exceeded HUCBC migration to normal heart homogenate. The HUCBC migration remained greatest at 2 and 24 h after LAD occlusion when the number of migrated cells was adjusted for the size of each myocardial infarction. Injection of 106 HUCBC in saline into infarcted myocardium of non immunosuppressed rats within 2 h (n=10) or at 24 h (n=5) after LAD occlusion resulted in infarction sizes 1 month later of 6.4 +/- 0.01% and 8.4 +/- 0.02% of the total left ventricular muscle area, respectively, in comparison with infarction sizes of 24.5 +/- 0.02% (n=10) in infarcted rat hearts treated with only saline (p < 0.005). Acute myocardial infarction in rats treated with only saline increased the myocardial concentration of tumor necrosis factor-alpha (TNF-alpha) from 6.9 +/- 0.8% to 51.3 +/- 4.6%, monocyte/macrophage chemoattractant protein (MCP-1) from 10.5 +/- 1.1% to 39.2 +/- 2.0%, monocyte inflammatory protein (MIP) from 10.6 +/- 1.6% to 23.1 +/- 1.5%, and interferon-gamma (INF-gamma) from 8.9 +/- 0.3% to 25.0 +/- 1.7% between 2 and 12 h after coronary occlusion in comparison with known controls (all p < 0.001). In contrast, the myocardial concentrations of these cytokines in rat hearts treated with HUCBC did not significantly change from the controls at 2, 6, 12, and 24 h after coronary occlusion. The present investigations suggest that infarcted myocardium significantly attracts HUCBC, that HUCBC can substantially reduce myocardial infarction size, and that HUCBC can limit the expression of TNF-alpha, MCP-1, MIP, and INF-gamma in acutely infarcted myocardium.