Transcripts regulated by the yeast nonsense-mediated and 5′ to 3′ mRNA decay pathways were identified by expression profiling of wild-type, upf1Δ, nmd2Δ, upf3Δ, dcp1Δ, and xrn1Δ cells. This analysis revealed that inactivation of Upf1p, Nmd2p, or Upf3p has identical effects on global RNA accumulation; inactivation of Dcp1p or Xrn1p exhibits both common and unique effects on global RNA accumulation but causes upregulation of only a small fraction of transcripts; and the majority of transcripts upregulated in upf/nmd strains are also upregulated to similar extents in dcp1Δ and xrn1Δ strains. Our results define the core transcripts regulated by NMD, identify several novel structural classes of NMD substrates, demonstrate that nonsense-containing mRNAs are primarily degraded by the 5′ to 3′ decay pathway even in the absence of functional NMD, and indicate that 3′ to 5′ decay, not 5′ to 3′ decay, may be the major mRNA decay activity in yeast cells.