The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.