Little is known about the molecules that mediate the attachment of proepicardial cells to the heart. Ephrins are cell surface ligands for Eph tyrosine kinase receptors, molecules known to play a role in cell adhesion and migration. Here, we detected EphrinB ligands in proepicardial and epicardial mesothelial cells (EMCs) using reverse transcriptase-polymerase chain reaction, immunoblotting, immunolocalization, and EphB-Fc binding. Aggregated EphB-Fc fragments clustered ephrinB1 ligands on living EMCs indicating that they are cell surface expressed. In vitro assays demonstrated that ephrinB ligands participate in EMC migration but not cell adhesion. Localization studies in hearts at Hamburger and Hamilton stage 30 and older revealed that ephrinB1 is expressed in the epicardium and subepicardial mesenchyme of the atrioventricular sulcus. EMCs treated with platelet-derived growth factor-BB expressed smooth muscle markers but not ephrinB1. Our study supports an early role for ephrinB ligands for migration of epicardial cells and a later role in perivascular fibroblasts of coronary vessels in the atrioventricular sulcus.