Phosphatidylglycerophosphate synthase catalyzes the committed step in the synthesis of the mitochondrial phospholipid cardiolipin. We showed previously that phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae is increased in conditions favoring mitochondrial development and during growth in the absence of inositol. Interestingly, the regulatory effects of inositol were not altered in ino2, ino4, or opi1 mutants suggesting that regulation in response to inositol is not at the level of gene transcription. We report here that steady state mRNA levels of the PGS1 gene, which encodes phosphatidylglycerophosphate synthase, were not altered by inositol or choline. Growth in the presence of the inositol-depleting drug valproate led to an increase in phosphatidylglycerophosphate synthase activity unaccompanied by increased PGS1 mRNA. PGS1 mRNA abundance was not decreased in ino2 or ino4 mutants and was unaffected in an opi1 mutant. Therefore, regulation of phosphatidylglycerophosphate synthase by inositol is not mediated at the level of mRNA abundance and does not require the INO2-INO4-OPI1 regulatory circuit. PGS1 was increased in glycerol/ethanol compared with glucose media and was maximally expressed as cells entered the stationary phase. Deletion of the mitochondrial genome did not affect PGS1 expression. Thus, whereas inositol controls phosphatidylglycerophosphate synthase activity, regulation of PGS1 expression occurs primarily in response to mitochondrial development cues.