Anti-tubulin antibodies secreted by plasmacytoma NSI-spleen cell hybrids were detected by an indirect binding assay. Different antibodies bound to different combinations of the tubulins as resolved by isoelectric focusing. Two monoclonal antibodies (TUB 2.1 and TUB 2.5) labeled only (i) the tubulin band on a polyacrylamide electropherogram and (ii) beta-tubulins as resolved by isoelectric focusing. The fraction that was specifically bound and eluted from antibody affinity columns was enriched in beta-tubulins as compared with alpha-tubulins, suggesting the possibility of some soluble tubulin homodimers and alpha,beta-heterodimers. Double labeling experiments were used to show that all detectable microtubules contained beta-tubulin.