In our previous study (Nishinaka et al., 1989), a chicken B cell line (HU3R27) deficient in thymidine kinase activity was used as a fusion partner for production of chicken monoclonal antibody. Specific antibody-producing hybridomas were initially obtained, but they soon lost the ability to produce antibody in culture. The present report documents the development of an improved fusion cell line. R27H4, for the production of chicken monoclonal antibody. This cell line was obtained by fusion of HU3R27 cells with spleen cells of immunized chicken. Fusion of R27H4 cells with spleen cells from chicken immunized by keyhole limpet hemocyanin (KLH) resulted in antibody-producing hybridomas. Subcloned hybridomas secreted highly reactive IgG and weakly reactive IgM. Some of the cell lines have been displaying stable antibody secretions for over 6 months.