To study the mechanism of selection of 5' splice sites, we first analyzed the in vitro time course of appearance of intermediates and products of splicing at a natural and at a cryptic 5' splice site. Our model system was a transcript derived from the early transcription unit 3 of adenovirus-2 harboring a cryptic 5' splice site Dcr1, 74 nucleotides downstream of the natural site D1. When studied in isolation, the two sites have different kinetics of splicing, Dcr1 being spliced markedly more slowly than D1. The upstream exon, shown elsewhere to have a positive effect on the selection of D1, has no influence on these kinetics; thus, it does not affect selection by modifying the kinetics of splicing. Nevertheless, this exon is of crucial importance for the exclusive selection of D1. We demonstrate that the cryptic site is recognized in all cases, but that exons harboring a potential stem-loop structure (HP1) prevent Dcr1 usage. The data suggest that the upstream exon sequences play the role of a cis-acting selector for the natural 5' splice site. The intrinsically rapid and efficient kinetics of splicing at the natural site and the selector function of the exon sequence may result in the exclusive use of the D1 site in the natural context.