Mutations in Drosophila alpha 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 alpha spectrin alleles (alpha-specrg41 and alpha-specrg35) on development and function of the larval midgut. Homozygous null alpha-specrg41-mutant larvae exhibited a striking defect in middle midgut acidification. In contrast, many homozygous alpha-specrg35 mutants were capable of acidification, indicating partial function of the truncated alpha-specrg35 product. Acidification was also blocked by a mutation in the labial gene, which is required for differentiation of cuprophilic cells in the midgut, suggesting that these cells secrete acid. We found that two isoforms of spectrin (alphabeta and alphabetaH) are segregated within the basolateral and apical domains of cuprophilic cells, respectively. The most conspicuous defect in cuprophilic cells from labial and alpha 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 alpha-specrg41 mutation in Drosophila interferes with the polarized activities of homologous molecules that drive acid secretion in cuprophilic cells.