Recently, the U.S. Environmental Protection Agency has supported the use of estrogenic screening assays for both mammalian and wildlife species in order to elucidate the contribution of environmental contaminants in the endocrine disrupter issue. Our lab has established an in vitro rainbow trout (Oncorhynchus mykiss) bioassay to screen for compounds that act through the rainbow trout estrogen receptor. The assay utilizes a recombinant chimeric receptor consisting of the Gal4-yeast DNA-binding domain linked upstream to the ligand-binding domain (A.A. 214-576) of the rainbow trout estrogen receptor (Gal4-RtDEF). This cDNA is co-transfected with a Gal4-regulated luciferase reporter gene that is expressed in the presence of ligand-bound Gal4-RtDEF. The transfection efficiency and performance of the Gal4-RtDEF and Gal4 reporter gene was evaluated in 3 fish cell lines, PLHC-1, RTG-2 and RTH-149 and the human breast cancer cell line, MCF7. A comparison of the ability of Gal4-RtDEF and the human chimeric Gal4-estrogen receptor (Gal-4HEG0) to bind 17b -estradiol (E2) in the MCF-7 cells revealed that the human receptor had approximately an order of magnitude greater sensitivity for E2 than the chimeric fish receptor. This was also reflected when two different fish cell lines, PLHC-1 and RTG-2, were transfected with either the fish or the human chimera. The PLHC-1 appeared to be the best cell line for transfection efficiency and inducibility; however, all three cell lines appeared to respond similarly when different estrogenic xenobiotics were added to the bioassay. This in vitro fish recombinant receptor bioassay is an excellent tool for fast, accurate, cost-effective screening of environmental contaminants or mixtures for estrogenic activities in fish.