Hepatocyte nuclear factors (HN-Fs) are upstream regulators of many liver-specific genes and are involved in many cellular functions in the body, but their existence, expression, and function in gonads are still poorly understood. Here we report on the first cloning of partial cDNAs of HNF-1 alpha and -1 beta and full HNF-3 beta cDNA from a tilapia (Oreochromis mossambicus) liver cDNA library. The deduced amino acid sequence of tilapia HNF-3 beta has a 90 to 96% identity with those of other fishes (dwarf gourami, medaka, and zebrafish), 75% with mammals (human, rat, and mouse), and 82% with Xenopus. RT-PCR detected IGF-I and -II and HNF-1 alpha, -1 beta, and -3 beta in both liver and gonads and the identity of the PCR fragments was confirmed by PCR hybridization. Immunoprecipitation and Western blotting also detected all three HNF proteins in both liver and gonads. Expression of HNFs in the gonads of the tilapia suggests that multi-HNFs may form a cascade to regulate gonadal physiology in the bony fish.