We constructed numerous deletion mutants in the 3' region of the yeast actin gene intron. When these were introduced into yeast on autonomously replicating shuttle vectors, a sequence element between 35 and 70 nucleotides upstream from the 3' splice site was required for splicing the actin gene transcripts. In mutant genes where this intron region was maintained but new sequences introduced immediately downstream replaced the normal 3' acceptor site sequences, alternative splicing occurred. The alternative splicing signal was generally the first AG dinucleotide following the intron sequence required for splicing. The intron-contained sequence is sufficient to define new intron-exon boundaries. It contains the octanucleotide 5'-TACTAACA-3', which occurs 20 to 55 nucleotides upstream from the 3' splice site in all split protein-coding nuclear genes from S. cerevisiae sequenced to date, and we suggest that it is an essential element of the yeast splicing mechanism, involved in the selection of splicing sites.