The evolutionarily conserved immune system of the zebrafish, in combination with its genetic tractability, position it as an excellent model system to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells, but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Utilizing regulatory elements of a Class II major histocompatibility gene, we generated a zebrafish reporter line expressing GFP in all antigen-presenting cells: macrophages, dendritic cells, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double transgenic animals demonstrated that kidney mhc2dab:GFP(hi); cd45:dsRed(hi) cells are exclusively mature monocytes/macrophages and dendritic cells as revealed by morphological and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs but were most abundant in the intestine and spleen, where they upregulate expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. Together, these findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.