Pluripotent embryonic stem (ES) cell cultures provide an efficient method for genome manipulation with many applications in marine biotechnology. To develop this technology we have been working to derive fish ES cell lines for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish. Zebrafish embryonal cell cultures were derived from blastula-stage embryos in LDF medium supplemented with fetal bovine serum, trout serum, trout embryo extract, selenium, insulin, and leukemia inhibitory factor. Cultures derived under these conditions on feeder layers of zebrafish embryonic fibroblasts possessed a diploid karyotype and exhibited an ES-like morphology with elevated levels of alkaline phosphatase enzyme activity. Injection of primary cell cultures derived from embryos of transgenic fish carrying neo produced chimeric fish detected by polymerase chain reaction analysis. Embryo cells cultured on poly-D-lysine substrate in the presence of retinoic acid or Buffalo rat liver cell-conditioned medium (BRL-CM) and a reduced serum concentration differentiated into neuronal cell types exhibiting elevated levels of acetylcholinesterase enzyme activity and expression of neurofilament and glial fibrillary acidic protein.