The majority of mutations that cause isolated GH deficiency type II affect splicing of GH1 transcripts, leading to the production of a dominant-negative GH isoform. Because numerous mutations and polymorphisms throughout the GH1 gene have not yet been tested for aberrant splicing, we used a deletion mutagenesis screen across intron 2-exon 3-intron 3 to identify splicing regulatory sequences. These analyses identified a new enhancer element, ESE2, upstream of the cryptic splice site in exon 3 and further defined a previously described enhancer (ESE1) to include the first seven nucleotides of exon 3. Besides enhancers, the overall size of intron 3 is also crucial for exon inclusion. Given the deleterious effects of the dominant-negative 17.5-kDa isoform, these and previous studies underscore the extent to which splicing regulatory elements serve to prevent exon skipping. Importantly, we show here that small interfering RNAs can be used to specifically degrade exon 3-skipped transcripts, potentially a new avenue of therapeutic intervention in isolated GH deficiency II and other dominant disorders.