The Tol2 transposon system can create chromosomal insertions in the zebrafish germ lineage very efficiently. We constructed a Tol2-based gene trap vector, T2KSAG, which contains a splice accepter, the GFP gene and the polyA signal. In the pilot screen for gene trapping using T2KSAG, we identified 38 fish lines expressing GFP in specific organs and tissues. In the SAGp53A line, GFP is expressed in the forebrain and midbrain, and the insertion of the gene trap construct captured a transcript of the kab gene encoding a zebrafish homolog of the human KARP (Ku86 autoantigen related protein)-binding protein (KAB). In the SAGm18B line, GFP is expressed in the central nervous system, and the insertion captured a transcript of a gene for succinyl CoA:3-oxoacid CoA-transferase (SCOT). Here, we describe how we performed the gene trap screen and characterized the gene trap insertions and will discuss the outcome of the pilot screen.