The US Environmental Protection Agency (USEPA) is faced with long lists of chemicals that require hazard assessment. The present study is part of a larger effort to develop in vitro assays and quantitative structure–activity relationships applicable to untested chemicals on USEPA inventories through study of estrogen receptor (ER) binding and estrogen-mediated gene expression in fish. The present effort investigates metabolic activation of chemicals resulting in increased estrogenicity. Phenolphthalin (PLIN) was shown not to bind rainbow trout (Oncorhynchus mykiss) ER (rtER) in a competitive binding assay, but vitellogenin (Vtg) expression was induced in trout liver slices exposed to 10^–4 and 10^–3.7 M PLIN. Phenolphthalein (PLEIN), a metabolite of PLIN, was subsequently determined to be formed when slices were exposed to PLIN. It binds rtER with a relative binding affinity to 17β-estradiol of 0.020%. Slices exposed to PLEIN expressed Vtg messenger RNA (mRNA) at 10^–4.3, 10^–4, and 10^–3.7 M, with no detectable PLIN present. Thus, Vtg expression noted in PLIN slice exposures was explained by metabolism to PLEIN in trout liver slices. A second model chemical, 4,4′-methylenedianiline (MDA), was not shown to bind rtER but did induce Vtg mRNA production in tissue slices at 10^–4.3, 10^–4, and 10^–3.7 M in amounts nearly equal to reference estradiol induction, thus indicating metabolic activation of MDA. A series of experiments were performed to identify a potential metabolite responsible for the observed increase in activity. Potential metabolites hydroxylamine-MDA, nitroso-MDA, azo-MDA, and azoxy-MDA were not observed. However, acetylated MDA was observed and tested in both ER-binding and tissue slice Vtg induction assays. Environ Toxicol Chem 2023;42:2747–2757. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.