The U1 snRNP is known to play a critical role in spliceosome assembly, at least in part through base pairing of its RNA moiety to the substrate, but many details remain to be elucidated. To further dissect U1 snRNA function, we have analyzed 14 single point mutations in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast Schizosaccharomyces pombe. Three of the four alleles previously found to support growth of Saccharomyces cerevisiae are lethal in S. pombe, implying a more critical role for the 5' end of U1 in fission yeast. Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site. The importance of U1 function in S. pombe is further underscored by the lethality of several single point mutants not examined previously in S. cerevisiae. In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth. Each viable mutant was tested for impaired splicing of three different S. pombe introns. Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor. Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site. Although point mutations at the 5' end of U1 have no discernible effect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment. Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast.