beta-Tubulins are encoded by members of multigene families and are generally highly conserved at the sequence level. The carboxyl terminal 15 amino acids are markedly more diverged than the rest of the sequence and constitute an "isotype defining region," which is conserved in corresponding beta-tubulin isoforms in different vertebrate species. It is thought that the carboxy terminus of beta-tubulin may not be required for assembly of microtubules per se, but it may be necessary for conferring properties on beta-tubulins required for isotype-specific functions. We have determined the extent to which a beta-tubulin isoform that lacks its carboxy terminus can assemble into functional suprastructures by generating two early-stop-codon variants of the gene for the testis-specific beta-tubulin (beta 2) in Drosophila melanogaster. We have also sequenced the null allele of this gene and discovered that it also contains an early-stop codon. By examining the products of these genes and the phenotypes they confer, we have determined that the beta-tubulin variants with large truncations (171 or 50 amino acids) do not accumulate to detectable levels and provide no beta-tubulin function. However, a small truncation missing only the terminal 15 amino acids is capable of being assembled into ultrastructurally normal looking microtubules in vivo, even though the truncated protein is less stable than wildtype beta 2. The functional failings of this truncated beta-tubulin are manifested in defective microtubule-based spermatogenic suprastructures, rather than at the level of assembly of individual microtubules. The most remarkable defect conferred by the truncated beta 2 is the failure of axonemes to assemble with proper organization, even though microtubules with presumptive axoneme identity are clearly present. We therefore demonstrate that the carboxy terminus of beta 2-tubulin is indeed required for organization of microtubule suprastructures in spermatogenesis. This observation supports the hypothesis that the variable carboxy terminus mediates isotype-specific microtubule-dependent functions.