During recent years invertebrates and especially molluscs have received increasing attention in the field of endocrine disruption and development of OECD test guidelines to assess the effects of endocrine disrupting compounds (EDCs) in molluscs is under development. The development of standardized tests to detect effects of EDCs in molluscs has proved cumbersome due to lack of specific biomarkers and endpoints for endocrine effects. Intersex (presence of oocytes in the testis) and induction of vitellogenin (the yolk protein precursor in oviparous vertebrates) have been used as biomarkers for EDCs in fish for decades. Vitellogenin (vtg) is mainly present in females, however, vtg synthesis can be induced by estrogens and EDCs in juveniles and males. During the last decade yolk protein has been used as biomarker in bivalve studies and alkali-labile phosphate (ALP) has been the applied method to indirectly estimate vtg levels. ALP was developed as an indirect method for determination of vtg in fish before more reliable and specific methods like ELISA (Enzyme-Linked Immuno Sorbent Assay) were developed. The use of yolk protein as biomarker in molluscs is based on the assumptions that vtg synthesis is also regulated by estrogens in molluscs even though it still remains unknown if and where vertebrate steroids are synthesized in molluscs and regulation of the endocrine system in molluscs is also unknown. By using our newly developed ELISA the present work investigates if yolk protein is a suitable biomarker for estrogenic exposure in molluscs by exposing the freshwater bivalve U. tumidus to 17β-estradiol (E2) (57, 164 and 512 ng/L) for eight weeks during their reproductive phase. Histological examination of the gonads revealed that E2 did not cause intersex and the ELISA revealed that the normal sex specific concentration distribution seen in fish was not seen in hemolymph of unexposed U. tumidus. The concentration of the protein did not differ among the sexes and was approximately 10000 times higher in male U. tumidus than in male fish. The results from the ELISA and ALP showed good correlation and the sites of yolk protein synthesis were investigated by immunohistochemistry. Based on the results we do not support the general use of yolk protein as biomarker for estrogenic exposure in bivalves because the yolk protein levels in unexposed males are high suggesting that yolk protein might have additional functions in U. tumidus.