SUMMARY:An enzyme immunoassay (EIA) system for kuruma prawn Penaeus japonicus vitellogenin (Vg) was developed and validated. In this assay, 96-well microtiter plates were coated with free vitellin (Vt). Assay conditions provided a detectable Vg range of 1.56–100 ng/well. The dose–response curve for serially diluted vitellogenic female hemolymph was parallel to the Vg standard curve. Male hemolymph produced absorbance values almost equal to background values, confirming the specificity of the assay. The intra- and interassay coefficient of variations were 6.0% and 10.5%, respectively. Using this assay, it was determined that very low levels of hemolymph Vg were detectable in prawns with oocytes at the previtellogenic stage. The Vg levels in the hemolymph rapidly increased at the endogenous vitellogenic stage and peaked at the early exogenous vitellogenic stage, but decreased at the late exogenous vitellogenic stage. Changes in hemolymph Vg levels preceded observed changes in gonadosomatic indices.