The epicardium of the mammalian heart arises from progenitor cells outside the developing heart. The epicardial progenitor (EPP) cells migrate onto the heart through a cyst-mediated mechanism in which the progenitors are released from the tissue of origin as cysts; the cysts float in the fluid of the pericardial cavity and attach to the naked myocardial surface of the heart, and cells in the cysts then migrate out to form an epithelial sheet. In this paper, we show that the gene encoding the alpha4 subunit of alpha4beta1 integrin (alpha4beta1) is essential for this migratory process. We have generated a knockin mutation in mice replacing the alpha4 integrin gene with the lacZ reporter gene, placing lacZ under the control of the alpha4 integrin promoter. We show that in homozygous mutant embryos, the migration of EPP progenitor cells is impaired due to inefficient budding of the cysts and a failure of the cells in the cysts to migrate on the heart. This study provides direct genetic evidence for essential roles for alpha4beta1 integrin-mediated cell adhesion in the migration of progenitor cells to form the epicardium, in addition to a previous finding that alpha4beta1 is essential for maintaining the epicardium (Yang, J.T., H. Rayburn, and R.O. Hynes. 1995. Development. 121:549-560).