The CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol:l-serine O-phosphatidyltransferase, EC 2.7.8.8) is one of the most highly regulated phospholipid biosynthetic enzymes in the yeast Saccharomyces cerevisiae. CHO1 expression is regulated by nutrient availability through a regulatory circuit involving a UAS(INO) cis-acting element in the CHO1 promoter, the positive transcription factors Ino2p and Ino4p, and the transcriptional repressor Opi1p. In this work, we examined the post-transcriptional regulation of CHO1 by mRNA stability. CHO1 mRNA was stabilized in mutants defective in deadenylation (ccr4Delta), mRNA decapping (dcp1), and the 5'-3'-exonuclease (xrn1), indicating that the CHO1 transcript is primarily degraded through the general 5'-3' mRNA decay pathway. In respiratory-sufficient cells, the CHO1 transcript was moderately stable with a half-life of 12 min. However, the CHO1 transcript was stabilized to a half-life of >45 min in respiratory-deficient (rho(-) and rho(o)) cells, the cox4Delta mutant defective in the cytochrome c oxidase, and wild type cells treated with KCN (a cytochrome c oxidase inhibitor). The increased CHO1 mRNA stability in response to respiratory deficiency caused increases in CHO1 mRNA abundance, phosphatidylserine synthase protein and activity, and the synthesis of phosphatidylserine in vivo. Respiratory deficiency also caused increases in the activities of CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, and the phospholipid methyltransferases. Phosphatidylinositol synthase and choline kinase activities were not affected by respiratory deficiency. This work advances our understanding of phosphatidylserine synthase regulation and underscores the importance of mitochondrial respiration to the regulation of phospholipid synthesis in S. cerevisiae.