We have developed a new exchangeable gene trap vector, pU‐17, carrying the intron‐lox71‐splicing acceptor (SA)‐βgeo‐loxP‐pA‐lox2272‐pSP73‐lox511. The SA contains three stop codons in‐frame with the ATG of βgalactosidase/neomycin‐resistance fusion gene (βgeo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre‐mutant lox system, we successfully replaced the βgeo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp‐deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the βgeo gene. Thus, using this pU‐17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain‐of‐function mutation by replacing the βgeo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre‐mediated recombination.