To study the effects and possible mechanisms of suspected endocrine disrupting compounds (EDCs), a wide variety of assays have been developed. In this work, we generated engineered Escherichia coli biosensor strains that incorporate the ligand-binding domains (LBDs) of the β-subtype estrogen receptors (ERβ) from Solea solea (sole), and Sus scrofa (pig). These strains indicate the presence of ligands for these receptors by changes in growth phenotype, and can differentiate agonist from antagonist and give a rough indication of binding affinity via dose-response curves. The resulting strains were compared with our previously reported Homo sapiens ERβ biosensor strain. In initial tests, all three of the strains correctly identified estrogenic test compounds with a high degree of certainly (Z′ typically greater than 0.5), including the weakly binding test compound bisphenol A (BPA) (Z′ ≈ 0.1–0.3). The modular design of the sensing element in this strain allows quick development of new species-based biosensors by simple LBD swapping, suggesting its use in initial comparative analysis of EDC impacts across multiple species. Interestingly, the growth phenotypes of the biosensor strains indicate similar binding for highly estrogenic control compounds, but suggest differences in ligand binding for more weakly binding EDCs. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.