A cloned rabbit beta-globin gene was modified by introducing a DNA fragment containing the 5' splice region of the large intron upstream or downstream of its natural counterpart. Analogous constructions were carried out with the 3' splice region. The genes were linked to SV40 DNA, transiently expressed in HeLa cells and the transcripts analyzed by S1 mapping. In all cases, the splice site further removed from the intron was utilized to the complete exclusion of its counterpart. This finding argues persuasively against a simple scanning model of RNA splicing, in which the splicing enzyme(s) attaches at the 'donor' spliced region and moves along the intron until it encounters the closest 'acceptor' splice region. A model compatible with the currently known facts is presented.