Most higher eukaryotic tubulins are separated into alpha- and beta-tubulin when electrophoresed in NaDodSO4- denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80). By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two alpha-tubulins and one beta-tubulin. Moreover, at least five alpha- and two beta-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Three of these alpha isotypes and one beta isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro. Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related. Possible mechanisms for the generation and selection of these tubulin isotypes are discussed.