A minor class of metazoan introns has well-conserved splice sites with 5'-AU-AC-3' boundaries, compared to the 5'-GU-AG-3' boundaries and degenerate splice sites of conventional introns. Splicing of the AT-AC intron 2 of a sodium channel (SCN4A) precursor messenger RNA in vitro did not require inhibition of conventional splicing and required adenosine triphosphate, magnesium, and U12 small nuclear RNA (snRNA). When exon 3 was followed by the 5' splice site from the downstream conventional intron, splicing of intron 2 was greatly stimulated. This effect was U1 snRNA-dependent, unlike the basal AT-AC splicing reaction. Therefore, U1-mediated exon definition interactions can coordinate the activities of major and minor spliceosomes.