Alternative splicing has been associated with increased evolutionary changes and with recent exon creation or loss. The addition of a new exon can be explained by its inclusion in only a fraction of the transcripts leaving the original form intact and giving to the new form the possibility to evolve independently but the exon loss phenomenon is less clear. To explore the mechanism that could be involved in CFTR exon 12 lower splicing efficiency in primates, we have analyzed the effect of multiple synonymous variations. Random patterns of synonymous variations were created in CFTR exon12 and the majority of them induced exon inclusion, suggesting a suboptimal splicing efficiency of the human gene. In addition, the effect of each single synonymous substitution on splicing is strongly dependent on the exonic context and does not correlate with available in silico exon splicing prediction programs. We propose that casual synonymous substitutions may lead to a reduced splicing efficiency that can result in a variable proportion of exon loss. If this phenomenon happens in in-frame exons and to an extent tolerated by the cells it can have an important evolutionary effect since it may generate a substrate for natural selection of new splicing isoforms.