The structural gene for a testis-specific beta--tubulin subunit in Drosophila melanogaster was mapped genetically and cytogenetically by means of a dominant male sterile mutation, B2tD, in which a variant form of the testis beta--tubulin is expressed. The B2t locus is at 48.5 map units on the third chromosome genetic map, and in bands 85D4-7 on the salivary chromosome map. The mutation B2tD causes disruption of microtubule function in all stages of spermatogenesis, beginning with meiosis. The effects of gene dosage of B2tD on meiosis were examined in detail cytologically at the light microscope level. In testes of flies in which the variant tubulin subunit is expressed, abnormal meiotic spindle formation, improper chromosome movement and failure to undergo cytokinesis occur. The extent of these defects in microtubule function depends on the dosage of the B2tD mutation, being most severe in males homozygous for the mutation, intermediate in males heterozygous for the mutation, and least marked in males heterozygous for B2tD and a tandem duplication of the region of the genome containing the B2t locus. Chromosomal events unrelated to microtubule function, such as replication and condensation, occur normally. Results obtained during mapping of the B2t locus strongly suggest a haplo-insufficient site at or closely linked to this locus.