An enhancer trap-based GAL4-UAS system in zebrafish requires strong GAL4 activators with minimal adverse effects. However, the activity of yeast GAL4 is too low in zebrafish, while a fusion protein of the GAL4 DNA-binding domain and the VP16 activation domain is toxic to embryonic development, even when expressed at low levels. To alleviate this toxicity, we developed variant GAL4 activators by fusing either multimeric forms of the VP16 minimal activation domain or the NF-κB activation domain to the GAL4 DNA-binding domain. These variant GAL4 activators are sufficiently innocuous and yet highly effective transactivators in developing zebrafish. Enhancer-trap vectors containing these GAL4 activators downstream of an appropriate weak promoter were randomly inserted into the zebrafish genome using the Sleeping Beauty transposon system. By the combination of these genetic elements, we have successfully developed enhancer trap lines that activate UAS-dependent reporter genes in a tissue-specific fashion that reflects trapped enhancer activities. Developmental Dynamics 238:641–655, 2009. © 2009 Wiley-Liss, Inc.