We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.