Pluripotent embryonic stem (ES) cells represent a cellular link between in vitro and in vivo genetic manipulation in animals and also have an enormous potential in development studies. To derive ES cells from the gilthead sea bream (Sparus aurata) species seems especially attractive due to its high economic value. To set up the methodology for feeder free cultivation, primary cultures were initiated from blastomeres, isolated from mid-blastula embryos. The media used were empirically designed according to Hong and Schartl (1996). Specific supplements were individually evaluated in terms of improving mitogenic activity and preventing cell differentiation. At present, putative ES cells, as suggested by their special morphology and by the positive staining to alkaline phosphatase, have been cultured for 6 passages. Chromosome analysis reveals a normal karyotype and high survival rate has been obtained subsequent to freezing. The next steps will consist of isolation of single colonies and additional testing of totipotency before being injected into recipient embryos to produce seabream chimeras.