The authors describe the development and characterization of rat monoclonal antibodies against tubulin using two different rat myeloma cell lines, Y3-Ag 1.2.3 and YB2/O. Both lines were used to produce hybridomas that secreted antitubulin antibodies, but YB2/O did not secrete myeloma light chains, making it a nonsecreting line. The antitubulin antibodies produced by these hybridomas showed high titers and were able to recognize tubulin from yeast, birds, and mammals. The antibodies were used for immunofluorescence staining of yeast mitotic spindles and interphase microtubule networks in tissue culture cells, with some differences in staining patterns observed between the two antibodies. The purified antibodies were conjugated to colloidal gold particles and used for electron microscopy. The study highlights the advantages of using rat × rat fusions for large-scale production of monoclonal antibodies and the stability of the YB2/O line. The antibodies were found to bind specifically to the α subunit of tubulin and showed potential for purifying microtubule-containing organelles.The authors describe the development and characterization of rat monoclonal antibodies against tubulin using two different rat myeloma cell lines, Y3-Ag 1.2.3 and YB2/O. Both lines were used to produce hybridomas that secreted antitubulin antibodies, but YB2/O did not secrete myeloma light chains, making it a nonsecreting line. The antitubulin antibodies produced by these hybridomas showed high titers and were able to recognize tubulin from yeast, birds, and mammals. The antibodies were used for immunofluorescence staining of yeast mitotic spindles and interphase microtubule networks in tissue culture cells, with some differences in staining patterns observed between the two antibodies. The purified antibodies were conjugated to colloidal gold particles and used for electron microscopy. The study highlights the advantages of using rat × rat fusions for large-scale production of monoclonal antibodies and the stability of the YB2/O line. The antibodies were found to bind specifically to the α subunit of tubulin and showed potential for purifying microtubule-containing organelles.