Estrogens control many physiological processes in both female and male vertebrates, mostly mediated by specific nuclear estrogen receptors (ER). Two ER subtypes (ERalpha and ERbeta) are present in most vertebrates, including the sea bream (Sparus auratus) a hermaphrodite teleost fish. In the present study several variant cDNAs encoding a second sea bream ERbeta (sbERbetab) is reported. Phylogenetic and Southern blot analysis indicate that sbERbetab and the previously cloned sbERbetaa (formerly sbERbeta) are encoded by different genes, which may have arisen by duplication of an ancestral ERbeta gene. Competitive binding assays show that sbERbetab has high affinity for 17beta-estradiol (K(d) = 1 nM) and specifically binds estrogen agonists (diethylstilbestrol and ethynylestradiol) and antagonists (ICI 182,780). In Northern blot sbERalpha, sbERbetaa, sbERbetab produce several different transcripts in a variety of tissues. RT-PCR showed a partially overlapping but differential tissue distribution in both male and female sea bream.