A new monoclonal antibody, specific to an epitope in the carboxyl terminus of the Drosophila collagen IV molecule (basement membrane collagen) was identified. The distributions of collagen IV, laminin, and an additional extracellular molecule, the 2G2 antigen (2G2-Ag), were followed immunocytochemically during early wing development. In late third instar larvae, collagen IV and laminin surround the entire wing disc, whereas the 2G2-Ag is limited to the region of the future wing pouch. For the first few hours following eversion of the disc, all three ECM components line the basal surfaces of all epithelial cells in the wing pouch, both those destined to line the wing veins and those destined to become tightly apposed in the large intervein regions. Collagen IV and laminin persist on these cells during the two initial rounds of apposition of dorsal and ventral wing surfaces; later, they become restricted to the cells lining the veins. The 2G2-Ag disappears completely quite early in the pupal period. Collagen IV appears to be synthesized at least twice, once in the larva and a second time in the pupa; in between it is enzymatically cleaved and may be eliminated, probably by hemocytes. In an extreme allele of blistered the wing is ballooned to form a single internal space. Collagen IV and laminin line all basal wing cell surfaces early in pupal development as they do in the wild type. Later, however, they continue to line the entire cavity of the mutant wing rather than assuming a restricted distribution. In a completely veinless wing (rhomboidveinletvein), collagen IV and laminin are also present generally on basal surfaces at early times, but are completely absent between the tightly apposed wing layers later. The ECM distributions both in wild type wings and in mutants suggest that the matrix plays a role in the establishment of the wing venation pattern. One possibility, strengthened by recent findings regarding ECM receptors in Drosophila, is their involvement in dorsal-ventral wing layer adhesion. Our findings also lead us to suggest that certain sets of features which distinguish vein from intervein cells may be linked during cell differentiation and thus help to define these cell phenotypes. The features include cytoskeletal specializations and certain cell surface and ECM molecules.