We have shown by means of conventional electron microscopy that the eggshell of Drosophila virilis at the main body of the laid egg consists of the vitelline membrane and the multilayered chorion, which includes the wax layer, the innermost chorionic layer, the endochorion, and the exochorion, while several specialized regions of the eggshell are seen across the anterior-posterior axis of the egg. Biochemical analysis revealed the existence of six quantitatively enriched chorion proteins. Among them, Dvs38 and Dvs36 are synthesized when the innermost chorionic layer and the endochorion are assembled. Immunogold electron microscopy has shown that these two proteins are incorporated in the morphologically complete vitelline membrane apparently through an intercalation process and represent structural components of the endochorion in all the specialized regions of the eggshell. Additionally, by cytochemical means, the enzyme eggshell peroxidase, which is synthesized in parallel with Dvs38 and Dvs36, has been identified as a structural component of the innermost chorionic layer and the endochorion. These findings suggest a complex protein-protein recognition pattern during the formation of the eggshell since the cosecretion of its components (i.e., Dvs38, Dvs36 chorion proteins and eggshell peroxidase) does not recommend their colocalization in the eggshell sublayers and the timing of their synthesis is not related to their final position on the eggshell (i.e., the identification of Dvs38 and Dvs36 chorion proteins as vitelline membrane components).