We are attempting to develop methods for in vitro culture of zebrafish embryonal stem cells. Primary cultures were initiated from wild-type zebrafish early embryos in basal nutrient medium supplemented with insulin, selenite, leukemia inhibitory factor, trout serum, fetal bovine serum, and trout embryo extract. In this medium, melanocytes appeared on the second day of culture. Basic fibroblast growth factor (bFGF) was mitogenic when cells were plated at low densities. bFGF suppressed melanogenesis is a dose-dependent fashion, with maximal effect at 20 ng/mL. Cultures initiated and maintained with bFGF for 24 hours and then incubated without bFGF for as long as 8 days did not contain pigmented cells. Experiments in which bFGF was added or removed at various times after initiation of cultures indicated that maximum sensitivity to bFGF occurred during the first 12 hours of culture. When wild-type cells from cultures without bFGF were injected into albino blastula-stage embryos, melanocytes subsequently developed in host embryos: no melanocytes appeared when cells from cultures with bFGF were injected into albino hosts.