The conserved nucleotides 9 and 10 of U1 small nuclear RNA (snRNA) have been proposed to base pair with either 5' exon or 3' splice site sequences. In S. pombe, U1 snRNA pairing with the conserved 3' splice site is required for the first step of splicing and viability. In contrast, we show that S. cerevisiae U1 mutants at positions 9 and 10 are fully functional. Splicing of several genes is normal in these strains, ruling out an essential base pairing between U1 snRNA and 3' splice sites. U1 snRNA positions 9 and 10 are shown to be involved in 5' splice site selection through their interaction with exon sequences. Our results demonstrate that some snRNA-pre-mRNA interactions are not evolutionarily conserved and that 3' splice site recognition occurs by different mechanisms in various organisms.