Three of the four alpha-tubulin genes in Drosophila melanogaster are temporally regulated. mRNA from one of these genes, alpha 85E-tubulin, first appears in 6- to 8-hr embryos and persists, with marked fluctuations, through the end of pupal development. In adults, alpha 85E mRNA has been unequivocally identified only in testes. In the present study, isotype-specific antibodies have been used to localize alpha 85E tubulin protein in whole tissues. The results demonstrate a spatially restricted expression pattern of the alpha 85E gene that includes tissues of both ectodermal and mesodermal origins. Specifically, embryonic accumulation of alpha 85E tubulin is limited to support cells of chordotonal organs and the developing musculature of the viscera and body wall. In late third instar larvae, chordotonal organs and a subset of larval nerves, but not muscle, stain with anti-alpha 85E. The timing of protein accumulation during pupal development suggests that alpha 85E tubulin is involved in the construction of the adult as well as the larval musculature. In testis, only the somatically derived cyst cells that surround developing spermatid bundles accumulate alpha 85E-tubulin. The cell types that express alpha 85E share a requirement for extensive cell shape changes during development, suggesting that this minor alpha-tubulin may have distinct functional properties.