In this study, an Ac-based gene trap construct was engineered to increase gene trapping efficiency by an effective use of triple acceptor sites preceding a reporter gene. The target of the engineering process was a synthetic intron preceding the GUS reporter. Two different gene trap constructs were designed. In one construct, three of the sequence elements serving as signals for recognition of an intron 3' boundary were systematically modified to allow for almost optimal acceptor site recognition, while these sequences remained unchanged in the other construct. To compare recognition of the engineered intron with that of the unmodified intron, tester constructs were transiently transformed into barley (Hordeum vulgare L.) tissue and the accuracy and efficiency of splicing was determined by mRNA mapping and reporter-gene expression frequency analysis. By employing this test system, we could show that systematic engineering of the intron sequence elements results in advanced intron recognition, compared to the unmodified intron, and that all three acceptor sites were activated, but with unequal frequency. The impact of our findings on reporter expression in a gene-trap approach is discussed.