Alternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with approximately 70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5' splice site located downstream of a weakened 5' splice site. We recovered two exonic and four intronic motifs that effectively silenced the proximal 5' splice site both in vitro and in vivo. Surprisingly, silencing was only observed in the presence of the competing upstream 5' splice site. Biochemical evidence strongly suggests that the silencing motifs function by altering the U1 snRNP/5' splice site complex in a manner that impairs commitment to specific splice site pairing. The data indicate that perturbations of non-rate-limiting step(s) in splicing can lead to dramatic shifts in splice site choice.