Five mouse a-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the a-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (Ma3 and Ma7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of a-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that (i) the duplication event leading to a pair of genes encoding a testis-specific ca-tubulin isotype predated the mammalian radiation, and (ii) both members of the duplicated sequence have been maintained since species divergence. A second ot-tubulin gene, Mca6, is expressed ubiquitously at a low level, whereas a third gene, MaL4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct a-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various at-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.Microtubules are assembled from heterodimers of a-and ,B-tubulin together with microtubule-associated proteins. They function in a wide variety of ways in eucaryotic cells; for example, they have specific functions in the mitotic and meiotic spindle, in the centriole, in the manchette and flagellar axoneme of spermatozoa, in axonal transport in neurons, and in the marginal bands of platelets. Clearly, associated proteins such as kinesin (28) play a crucial role in conferring these and other specific functions on microtubules. Additionally, a-and f-tubulin proteins themselves show significant heterogeneity, and the tubulin isotypes described to date vary in their patterns of expression in an evolutionarily conserved manner (16). This heterogeneity in a-and ,-tubulins offers the potential of contributing to diversity of microtubule function in eucaryotic cells, either through differential polymerization of the various tubulin subunits, or by virtue of unique interaction(s) with associated proteins.In mammals, a-and ,B-tubulins are encoded by large multigene families (5). A significant fraction of the genes in these families are pseudogenes (5, 13), a fact that makes it difficult to distinguish functional from nonfunctional genomic tubulin sequences. To circumvent this problem and to study the important question of the tubulin repertoire of mammals, we decided to study expressed mouse tubulin genes at the mRNA level by exhaustive screening of cDNA libraries. We re...