) was constructed to investigate the function of immune-related proteins and to gain insight into their mechanisms of interaction. The titer of this original library was 1.6×10⁶ CFU/ml. Most of the fragments PCR-amplified from single colonies were larger than 500 bp, with an average length of 1.5 kb. We randomly selected and sequenced 115 expressed sequence tags. The capacity of the amplified library was approximately 1.8×10¹² CFU. After transformation into yeast cells, the transformation efficiency was 3.6×10⁵ CFU/μg, and the capacity was 5.4×10⁶ CFU. The successful construction of this yeast two-hybrid library provides an effective tool to elucidate mechanisms of immunity, especially signaling pathways, in this flounder species.