Nonsense-mediated decay (NMD) rids eukaryotic cells of aberrant mRNAs containing premature termination codons. These are discriminated from true termination codons by downstream cis-elements, such as exon-exon junctions. We describe three novel human proteins involved in NMD, hUpf2, hUpf3a, and hUpf3b. While in HeLa cell extracts these proteins are complexed with hUpf1, in intact cells hUpf3a and hUpf3b are nucleocytoplasmic shuttling proteins, hUpf2 is perinuclear, and hUpf1 cytoplasmic. hUpf3a and hUpf3b associate selectively with spliced beta-globin mRNA in vivo, and tethering of any hUpf protein to the 3'UTR of beta-globin mRNA elicits NMD. These data suggest that assembly of a dynamic hUpf complex initiates in the nucleus at mRNA exon-exon junctions and triggers NMD in the cytoplasm when recognized downstream of a translation termination site.