Two independent transcriptional activation functions have been mapped to the N- and C-terminal domains of estrogen receptors (ERs), and are named activation function-1 (AF-1) and AF-2, respectively. Due to the lower activity of AF-1 and difficulties in producing AF-1 recombinant protein, little information is available regarding the biochemical properties of ER AF-1 and its coactivators compared to AF-2. In this study, we characterized the AF domains from medaka fish ER alpha (meER alpha) using a transient expression assay in cultured mammalian cells. While both meER alpha AF-1 and AF-2 were functional and gave similar results to human ER alpha AFs, meER alpha AF-1 displayed significant activity even in HeLa cells that exhibit little human ER alpha (hER alpha) AF-1 activity. Evidence of transcriptional squelching between hER alpha and meER alpha AF-1 molecules suggested that the molecules utilized common coactivators in mammalian cells. We also showed that large amounts of the meER alpha A/B domain could be expressed in Escherichia coli cells as a soluble protein, in contrast to hER alpha A/B domain protein which was not observed. Taken together, our results suggested that meER alpha AF-1 may have a more significant role in estrogen-induced function of meER alpha than AF-2 in medaka fish.