The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform ,3-tubulin, the wild-type testis-specific isoform 132-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B218. The testis-specific j2-tubulin is highly homologous to the major vertebrate beta-tubulins, but j3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for 02-and 03-tubulin are not the satne. The structure of the gene for the variant ,3-tubulin isoform, but not that of the testis-specific 112-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B218 in the gene for the testis-specific ,2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B218 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila 12-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.