Targeted gene expression is a powerful tool for understanding gene function in vivo. In zebrafish, over-expression of gene products is typically accomplished ubiquitously, without temporal and spatial specificity. However, the yeast Gal4/UAS system can be used for targeted gene expression in zebrafish. Here we describe the generation and characterization of Tg[gsc: Gal4-VP16] transgenic zebrafish lines that harbor a construct encoding Gal4-VP16 transcriptional activator under the control of a fragment of the goosecoid gene promoter. Tg[gsc:Gal4-VP16] embryos express Gal4-VP16 RNA in presumptive prechordal plate mesendoderm during late blastula and throughout gastrulation. By crossing these fish to Tg[UAS-GFP] transgenic fish, we show that the gsc:Gal4-VP16 transgene is capable of driving strong expression of a target gene in the prechordal plate and its derivatives during gastrulation and segmentation. Thus, the use of Tg[gsc:Gal4-VP16] fish can help in understanding gene function in the prechordal plate, an embryonic structure that is crucial for normal neural patterning.