Nonsense-mediated mRNA decay (NMD) represents a phylogenetically widely conserved splicing- and translation-dependent mechanism that eliminates transcripts with premature translation stop codons and suppresses the accumulation of C-terminally truncated peptides. Elimination of frameshifted transcripts that result from faulty splicing may be an important function of NMD. To test this hypothesis directly, this study used the IVS1 + 5 G>A thalassemia mutation of the human beta-globin gene as a model system. We generated beta-globin gene constructs with this mutation and an iron-responsive element in the 5' untranslated region, which allowed specific experimental activation and inactivation of translation and, hence, NMD of this transcript. Premessenger RNAs with IVS1 + 5 G>A were spliced at normal sites and cryptic sites, enabling a direct comparison of the effect of NMD on the accumulation of normal and frameshifted messenger RNAs. In transfected HeLa cells, the predominant frameshifted transcript was degraded under conditions of active NMD, whereas accumulation to high levels occurred under conditions of specifically disabled NMD, thereby indicating an important physiologic function of NMD in the control of the splicing process. An unexpected finding was that accumulation of a second aberrant transcript remained unaffected by NMD. The IVS1 + 5 G>A mutation thus revealed the presence of an unknown cis-acting determinant that influences the NMD sensitivity of a putative NMD substrate. It can therefore serve as a useful tool for defining the mechanisms that permit specific transcripts to circumvent the NMD pathway.