The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.