In vivo analyses of cis-acting sequence requirements for pre-mRNA splicing in tobacco nuclei have previously demonstrated that the 5' splice sites are selected by their position relative to AU-rich elements within plant introns and by their degree of complementarity to the U1 small nuclear RNA. To determine whether the presence of adjacent introns affects 5' splice site recognition in plant nuclei, we have analyzed the in vivo splicing patterns of two-intron constructs containing 5' splice site mutations in the second intron. These experiments indicated that the splice site selection patterns in plant nuclei are defined primarily by sequences within the intron (intron definition) and secondarily by weak interactions across exons (exon definition). The effects of these secondary interactions became evident only when mutations in the downstream 5' splice site decreased its functionality and differed depending on the availability of cryptic splice sites close to the mutant site. In beta-conglycinin chimeric transcripts containing multiple cryptic 5' splice sites, the presence of an intact upstream intron significantly increased splicing at the downstream 5' splice sites in a polar fashion without activating exon skipping. In a natural beta-conglycinin transcript, which does not contain cryptic 5'splice sites, mutation of the first nucleotide of the downstream intron activated an array of noncanonical 5' and 3' splice sites and some exon skipping.