Mature tRNA is normally extensively modified and extremely stable. Recent evidence suggests that hypomodified mature tRNA in yeast can undergo a quality control check by a rapid tRNA decay (RTD) pathway, since mature tRNA(Val(AAC)) lacking 7-methylguanosine and 5-methylcytidine is rapidly degraded and deacylated at 37 degrees C in a trm8-Delta trm4-Delta strain, resulting in temperature-sensitive growth. We show here that components of this RTD pathway include the 5'-3' exonucleases Rat1 and Xrn1, and Met22, which likely acts indirectly through Rat1 and Xrn1. Since deletion of MET22 or mutation of RAT1 and XRN1 prevent both degradation and deacylation of mature tRNA(Val(AAC)) in a trm8-Delta trm4-Delta strain and result in healthy growth at 37 degrees C, hypomodified tRNA(Val(AAC)) is at least partially functional and structurally intact under these conditions. The integrity of multiple mature tRNA species is subject to surveillance by the RTD pathway, since mutations in this pathway also prevent degradation of at least three other mature tRNAs lacking other combinations of modifications. The RTD pathway is the first to be implicated in the turnover of mature RNA species from the class of stable RNAs. These results and the results of others demonstrate that tRNA, like mRNA, is subject to multiple quality control steps.