The endothelium of the embryonic vertebrate heart evokes a regional specificity that remains an unexplained phenomenon in cardiac morphogenesis. A restricted population of endothelial cells lining the atrioventricular (AV) canal and proximal outflow tract (OT) transforms into mesenchyme, the reputed progenitor of the valves and membranous septa. The remainder of the cells lining these and other regions of the heart, in particular the ventricle, stay epithelial. At the present time there is no information regarding the determinants for endothelial cell diversity. To investigate the molecular basis for functionally distinct endothelial cell populations, we undertook a search for cell surface proteins within the endocardial cushions of Day 4 chicken embryos that might be sensitive to subtle differences in endothelial cell composition. We theorized that monoclonal antibodies raised against proteins expressed during early valve morphogenesis could provide markers for endothelial subpopulations, thereby assisting our efforts in defining, and determining the origin of, endothelial heterogeneity. In the present study, an in vitro collagen gel culture assay was employed to identify an antibody, JB3, that distinguishes between AV/OT endothelium and ventricular endothelium. Based on this assay, JB3-positive material was associated only with AV/OT endothelia or the mesenchyme derived from these epithelia. Also, a network of JB3-positive fibrillar material was observed within the collagen gel surrounding the explanted cells. The JB3 antigen showed a conspicuous distribution in pregastrulation-stage chicken embryos with immunolabeling observed in the initial primitive streak at 5 hr incubation (stage 2). Subsequent detection in the definitive primitive streak, Hensen's node, and notochord indicate a consistent relationship to midline structures. JB3 antigen also localized to the regions of presumptive precardiac mesoderm and, at later stages, neural crest, somites, and ventral mesocardium. These data suggest that the JB3 antigen may play a role in establishing cardiac endothelial diversity by defining a subpopulation of cells destined to participate in valve formation. Moreover, JB3 may also influence formation of the primary axis and mesoderm structures that form at the midline. Immunochemical analyses showed that JB3 recognizes a polypeptide that migrates near the molecular weight position of fibrillin (350-390 kDa), the extracellular matrix protein linked to the Marfan syndrome. Based on the molecular mass and similar immunostaining patterns in early embryos, we propose that the JB3 antigen is a fibrillin isotype or a fibrillin-associated protein.