In order to analyze migration patterns of mouse neural crest cells, we produced a rat anti-mouse monoclonal antibody (4E9R) which identifies these cells. The distribution of 4E9R-immunoreactive cells was examined in migratory stages of mouse neural crest cells, ranging from embryonic day (Ed) 8.5 to 10.5. In the cephalic region of Ed 8.5 embryos, some mesencephalic mesenchymal cells close to neural folds were 4E9R-positive. In Ed 9.0-9.5 embryos, streams of 4E9R-immunoreactive cells extending from cranial neural tubes to maxillary processes and to first visceral arches were found in the lateral region and some of these cells were localized in presumptive trigeminal ganglia. In the same embryonic stages, 4E9R-positive cells were present in mesenchymal cells around the optic and otic vesicles. In the trunk region of Ed 9.5-10.5 embryos, 4E9R-positive cell masses were observed in dorsolateral spaces adjacent to neural tubes. The presence of 4E9R-immunoreactive cells in somitic segments was restricted within the anterior halves and these cells were seen under the dermomyotome and/or in the medial portion of the sclerotome. These cells colonized in presumptive dorsal root ganglia and in the surroundings of the dorsal aorta, the embryonic area in which sympathetic ganglia are formed. 4E9R-positive cells were also found just under the epidermis. These observations indicate that the distribution of 4E9R-positive cells is similar to that of amniote neural crest cells reported previously. Furthermore, the data suggest that the migratory behavior of mouse neural crest cells at trunk levels may be different between rostral and caudal levels within an anterior half of the sclerotome, and that sympathetic ganglia may be formed by neural crest cells migrating along "ventromedial pathways" as well as "ventrolateral pathways" at hindlimb-bud levels of mouse embryos. In primary cultures of mouse neural crest cells, approximately 80% of the cells were 4E9R-positive on culture day 2. Further, we have shown in cultures treated with colchicine or cytochalasin B that 4E9R antigens are present in intermediate filaments. When image analysis with a confocal laser scanning microscope was performed on primary cultures of mouse neural crest cells, the intracellular localization of 4E9R antigens in these cells was comparable to that of vimentin, which is a typical intermediate filament in most mesenchymal cells in embryonic stages examined. Since the distribution of 4E9R-positive cells and anti-vimentin-immunoreactive cells was different in mouse embryos, it is suggested that 4E9R antigens are vimentin-related and specifically expressed in a few cell types including mouse neural crest cells. These results indicate that a rat anti-mouse monoclonal antibody 4E9R is useful for the identification of mouse neural crest cells during the migratory stages.