The eggshell in Drosophila is produced by ovarian follicle cells during the later stages of oogenesis. Eggshell formation involves the ordered synthesis and assembly of several protein components. Genes encoding the most abundant eggshell proteins have been identified by molecular cloning studies. Morphological examination of eggs produced by females carrying female sterile mutations on the X and third chromosomes have revealed additional loci involved in chorion formation. In this study we screened a collection of female sterile mutants carrying EMS-induced mutations on the second chromosome for eggshell mutants. A class of six mutants with potential vitelline membrane defects was identified on the basis of the response of mutant eggs to hypochlorite solutions. Biochemical analysis showed that one mutant, fs(2)QJ42, failed to produce a major vitelline membrane protein, sV23. The mutation was mapped cytogenetically to 26A, a region previously implicated in vitelline membrane formation by molecular cloning studies. Northern blot analysis using a cloned copy of the sV23 gene as probe showed a 10- to 15-fold reduction of sV23 RNA levels in the mutant. sV23 synthesis and fertility were restored when a normal copy of the sV23 gene was introduced into the mutant via germ line transformation. Transposons carrying the sV23 gene with as little as 147 bp of 5' flanking DNA were capable of restoring fertility and sV23 protein to wild type levels.