During vertebrate embryogenesis, interaction between neural crest cells and the enteric mesenchyme gives rise to the development of the enteric nervous system. In birds, monoclonal antibody HNK-1 is a marker for neural crest cells from the entire rostrocaudal axis. In this study, we aimed to characterize the HNK-1 carrying cells and antigen(s) during the formation of the enteric nervous system in the hindgut. Immunohistological findings showed that HNK-1-positive mesenchymal cells are present in the gut prior to neural crest cell colonization. After neural crest cell colonization this cell type cannot be visualized anymore with the HNK-1 antibody. We characterized the HNK-1 antigens that are present before and after neural crest cell colonization of the hindgut. Immunoblot analysis of plasma membranes from embryonic hindgut revealed a wide array of HNK-1-carrying glycoproteins. We found that two HNK-1 antigens are present in E4 hindgut prior to neural crest cell colonization and that the expression of these antigens disappears after neural crest colonization. These two membrane glycoproteins, G-42 and G-44, have relative molecular masses of 42,000 and 44,000, respectively, and they both have isoelectric points of 5.5 under reducing conditions. We suggest that these HNK-1 antigens and the HNK-1-positive mesenchymal cells have some role in the formation of the enteric nervous system.