Tandem donor splice sites (5 ' ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Delta 3 tandem 5 ' ss are a specific subclass of 5 ' ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5 ' ss is typically preferred, yet factors governing particular 5 ' ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Delta 3 tandem 5 ' ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5 ' ss. However, the downstream 5 ' ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5 ' ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5 ' ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5 ' ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5 ' ss interaction is more flexible than previously thought.