One of the major changes that occurs during the maturation of oocytes is the accumulation of yolk protein, or vitellin (Vn). To better understand how this process is regulated, we characterized the Vn of the ridgeback shrimp, Sicyonia ingentis (Penaeoidea). This Vn is a 322 kDa molecule composed of three subunits. Using purified Vn, we developed an anti-Vn antiserum and used it to characterize vitellogenin by Western blot analysis. The antiserum was also used in an ELISA to measure hemolymph levels of vitellogenin. Previous studies suggested the presence of vertebrate-type steroids might stimulate reproductive processes in decapod crustaceans. Treatment of sexually quiescent female shrimp with progesterone, hydroxyprogesterone, and estradiol did not increase hemolymph levels of yolk protein precursor. The absence of a response to these steroids may reflect the presence of other hormones (such as the gonad-inhibiting hormone) that prevent oocyte development. To examine the molecular basis for the regulation of vitellogenesis, ovarian and hepatopancreas expression cDNA libraries were screened using the anti-Vn antiserum. A 2.9 kilobase clone was isolated from both cDNA libraries suggesting that both tissues are sites of vitellogenin synthesis. These molecular tools should be useful for in vitro studies of vitellogenin synthesis.