Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.