Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 59 splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 59 pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 59 splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 59 splice site, rather than repressing the 39 following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 59 pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 59 splice site follows a scanning-type mechanism, precluding competition with other functional 59 pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 59 pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 59À39oriented scanning process for accurate recognition of the authentic 59 splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 59 splice site selection.