The Fish Sexual Development Test (FSDT) has gone through two validations as an OECD test guideline for the detection of endocrine active chemicals with different modes of action. The validation has been finalized on four species: Zebrafish (Danio rerio), Japanese medaka (Oryzias latipes), three spined stickleback (Gasterosteus aculeatus) and fathead minnow (Pimephales promelas) and with four model compounds: the weak estrogens 4-tert Octylphenol and 4-tert pentylphenol, the aromatase inhibitor prochloraz and the androgen dihydrotestosterone. All species were tested in different laboratories to facilitate inter laboratory comparison. A NOEC-design with three exposure concentrations in four replicates was applied to all species. Flow through exposure to chemicals began with fertilised eggs and continued until 60 days post hatch. At sampling, the fish were either separated in three parts: A tail and a head part, which were pooled and homogenised for vitellogenin analysis and a body part that were fixed for histological evaluation or a blood sample were taken for vitellogenin analysis and the body were fixed for histology or the liver were sampled for vitellogenin analysis and the body were fixed for histology. For all three methods, the fish parts were numbered and histology could therefore be linked to the vitellogenin concentration in individual fish. The two core endocrine relevant endpoints were vitellogenin concentrations and phenotypic sex ratio. Change in the sex ratio is presented as a population relevant endpoint and the results of the two validation rounds will be discussed in relation to environmental risk assessment and species selection.