Abstract Mutations in Drosophilaα spectrin cause larval lethality and defects in cell shape and adhesion (J. Lee et al.,1993, J. Cell Biol.123, 1797–1809). Here we examined the effects of two lethal α spectrin alleles (α- spec rg41 and α- spec rg35 ) on development and function of the larval midgut. Homozygous null α- spec rg41 -mutant larvae exhibited a striking defect in middle midgut acidification. In contrast, many homozygous α- spec rg35 mutants were capable of acidification, indicating partial function of the truncated α- spec rg35 product. Acidification was also blocked by a mutation in the labialgene, which is required for differentiation of cuprophilic cells in the midgut, suggesting that these cells secrete acid. We found that two isoforms of spectrin (αβ and αβ H) are segregated within the basolateral and apical domains of cuprophilic cells, respectively. The most conspicuous defect in cuprophilic cells from labialand α spectrin mutants was in morphogenesis of the invaginated apical domain, although basolateral defects may also contribute to the acidification phenotype. Acid secretion in vertebrate systems is thought to involve the polarized activities of apical proton pumps and basolateral anion exchangers, both of which interact with spectrin. We propose that the α- spec rg41 mutation in Drosophilainterferes with the polarized activities of homologous molecules that drive acid secretion in cuprophilic cells.