Variations at position +3 of 5' splice-sites (5'ss) are reported to induce aberrant splicing in some cases but not in others suggesting that the overall nucleotidic environment can dictate the extent to which 5'ss are correctly selected. Functional studies of three variations identified in donor splice-sites of USH2A and PCDH15 genes sustain this assumption. To gain insights into this question, we compared the nucleotidic context of U2-dependent 5'ss naturally deviated (+3G, +3C, or +3T) from the +3A consensus with 5'ss for which a +3 variation (A>G, A>C, or A>T) was shown to induce aberrant splicing. Statistical differences were found between the two datasets, highlighting the role of one peculiar position in each context (+3G/+4A; +3C/-1G; and +3T/-1G). We provided experimental support to the biostatistical results through the analysis of a series of artificial mutants in reporter minigenes. Moreover, different 5' end-mutated U1 snRNA expression plasmids were used to investigate the importance of the position +3 and of the two identified compensatory positions -1 and +4 in the recognition of 5'ss by the U1 snRNP Overall, our findings establish general properties useful to Molecular geneticists to identify nucleotide substitutions at position +3 that are more likely to alter splicing