The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers an intracellular signaling pathway, the unfolded protein response (UPR), that leads to increased transcription of genes encoding ER-resident proteins. Transcriptional activation is mediated by a dedicated transcription factor, Hac1p, whose activity is controlled by regulated splicing of its mRNA. We have identified a mutation in tRNA ligase that disrupts the UPR in the yeast Saccharomyces cerevisiae. In this mutant, splicing of HAC1 mRNA, but not tRNA, is blocked. In contrast, HAC1 mRNA splicing is not impaired in cells that are blocked in spliceosome-mediated mRNA splicing. Furthermore, the splice junctions of HAC1 mRNA do not conform to the consensus sequences of other yeast pre-mRNAs. Our results suggest that the regulated splicing of HAC1 mRNA occurs by a novel pathway, involving tRNA ligase and bypassing the spliceosome.