Rat monoclonal antitubulin antibodies were developed using a new nonsecreting rat myeloma cell line. Hybrid myeloma cell lines were created by fusing rat myelomas with spleen cells from rats immunized with yeast tubulin. Two cell lines, Y3-Ag 1.2.3. and YB2/O, were compared. YB2/O, a nonsecreting line, produced hybrids without myeloma chain components, making it a better parental line for monoclonal antibody production. The resulting antibodies recognized tubulin from yeast, birds, and mammals and showed clear immunofluorescent staining of yeast mitotic spindles and interphase microtubules in tissue culture cells. The purified antibodies could be conjugated to colloidal gold particles for electron microscopy. Antitubulin antibodies are important for studying microtubule structure and function in cells. They are used in immunofluorescence to examine microtubule responses to stimuli like drugs or calcium ions. Electron microscopy further extends structural information from immunofluorescence. These antibodies can also be used to purify microtubule-containing organelles. However, tubulin is a poor antigen, and antisera have low titers, making monoclonal antibodies more suitable. The new YB2/O line allows for unlimited monoclonal antibody production, which is more convenient than current monospecific antisera. The YB2/O line was derived from a hybrid myeloma and is nonsecreting, making it suitable for producing monoclonal antibodies. It was used in fusion experiments with Y3-Ag 1.2.3. and YB2/O to produce antitubulin monoclonal antibodies. The hybrids produced by YB2/O did not express the myeloma light chain, making them more suitable for immunofluorescence. The antibodies were purified from serum and ascites, and their properties were analyzed. They showed high binding activity to tubulin and were used for immunofluorescence and electron microscopy. The results indicated that the antibodies were of the IgG class and had high specificity for tubulin. The antibodies were also used to stain yeast and tissue culture cells, showing clear microtubule staining. The study demonstrated the effectiveness of the YB2/O line in producing high-quality monoclonal antibodies for research purposes.Rat monoclonal antitubulin antibodies were developed using a new nonsecreting rat myeloma cell line. Hybrid myeloma cell lines were created by fusing rat myelomas with spleen cells from rats immunized with yeast tubulin. Two cell lines, Y3-Ag 1.2.3. and YB2/O, were compared. YB2/O, a nonsecreting line, produced hybrids without myeloma chain components, making it a better parental line for monoclonal antibody production. The resulting antibodies recognized tubulin from yeast, birds, and mammals and showed clear immunofluorescent staining of yeast mitotic spindles and interphase microtubules in tissue culture cells. The purified antibodies could be conjugated to colloidal gold particles for electron microscopy. Antitubulin antibodies are important for studying microtubule structure and function in cells. They are used in immunofluorescence to examine microtubule responses to stimuli like drugs or calcium ions. Electron microscopy further extends structural information from immunofluorescence. These antibodies can also be used to purify microtubule-containing organelles. However, tubulin is a poor antigen, and antisera have low titers, making monoclonal antibodies more suitable. The new YB2/O line allows for unlimited monoclonal antibody production, which is more convenient than current monospecific antisera. The YB2/O line was derived from a hybrid myeloma and is nonsecreting, making it suitable for producing monoclonal antibodies. It was used in fusion experiments with Y3-Ag 1.2.3. and YB2/O to produce antitubulin monoclonal antibodies. The hybrids produced by YB2/O did not express the myeloma light chain, making them more suitable for immunofluorescence. The antibodies were purified from serum and ascites, and their properties were analyzed. They showed high binding activity to tubulin and were used for immunofluorescence and electron microscopy. The results indicated that the antibodies were of the IgG class and had high specificity for tubulin. The antibodies were also used to stain yeast and tissue culture cells, showing clear microtubule staining. The study demonstrated the effectiveness of the YB2/O line in producing high-quality monoclonal antibodies for research purposes.