This Test Guideline describes an in vivo screening assay for certain endocrine active substances where sexually mature male and spawning female fish are held together and exposed to a chemical during a limited part of their life-cycle (21 days). This assay covers the screening of oestrogenic and androgenic activity, and aromatase inhibition. The assay was validated on the fathead minnow (Pimephales promelas), the Japanese medaka (Oryzias latipes) and the zebrafish (Danio rerio); however zebrafish does not allow the detection of androgenic activity. At termination of the 21-day exposure period, depending on the species used, one or two biomarker endpoint(s) are measured in males and females as indicators of oestrogenic, aromatase inhibition or androgenic activity of the test chemical; these endpoints are vitellogenin and secondary sexual characteristics. Vitellogenin is measured in fathead minnow, Japanese medaka and zebrafish, whereas secondary sex characteristics are measured in fathead minnow and Japanese medaka only.