Blood cells of the colonial tunicate Botryllus were separated by density gradient centrifugation in Percoll. Unseparated blood cells were used to immunize mice for development of hybridoma cell lines producing anti-Botryllus monoclonal antibodies. These antibodies identify specific subpopulations of blood cells, indicating possible divisions of these cells into defined subgroups sharing particular differentiation antigens. Additional studies utilizing fluorescein-conjugated lectins also revealed differential binding to density- and monoclonal antibody-defined blood cell fractions. These methods allow separation of the different Botryllus blood cell types for functional studies.