While several mutagenesis methods have been successfully applied in zebrafish, these mutations do not allow tissue- or temporal-specific functional analysis. We have developed a strategy that will allow tissue- or temporal-specific disruption of genes in zebrafish. This strategy combines gene-trap mutagenesis and FlEx modules containing target sites for site-specific recombinases. The gene-trap cassette is highly mutagenic in one orientation and nonmutagenic in the opposite orientation, with different fluorescent proteins as indicators of the orientation. The inclusion of the FlEx modules allows two rounds of stable inversion mediated by the Cre and Flp recombinases. This gene-trap cassette can be easily delivered via transposons. Through large-scale community-wide efforts, broad genome coverage can be obtained. This should allow investigation of cell/tissue-specific gene function of a wide range of genes.