We report the construction, characterization, and use of luciferase reporters to test the ability of antisense oligonucleotides to inhibit RNA splicing. beta-Globin and adenovirus introns were inserted into a luciferase cDNA, and luciferase expression was analyzed in transiently transfected cells. The adenovirus reporter expressed large amounts of luciferase, but two beta-globin constructs were inactive. RNA analyses determined that the beta-globin pre-mRNAs were not spliced. Mutagenesis of the beta-globin 5' splice site, branchpoint, and 3' splice site sequences to the adenovirus intron sequences promoted maximal splicing and luciferase activity; reciprocal changes in all three elements of the adenovirus intron eliminated luciferase activity. Wild-type and 3' splice site mutated adenovirus reporters were used to determine the ability of phosphorothioate deoxy and 2' methoxy oligonucleotides to inhibit splicing. RNase H activating oligodeoxynucleotides were better inhibitors of wild-type adenovirus expression than were 2' methoxy analogues. However, 2' methoxy oligonucleotides specific for the branchpoint were more effective inhibitors of splicing of adenovirus transcript containing the beta-globin branchpoint and 3' splice site. We suggest that pre-mRNAs with weak splice sites are potential targets for oligonucleotides that inhibit splicing by occupancy rather than cleavage of the transcripts.