Abstract: In Saccharomyces cerevisiae, genes encoding phospholipid-synthesizing enzymes are regulated by inositol and choline (IC). The current model suggests that when these precursors become limiting, the transcriptional complex Ino2p-Ino4p activates the expression of these genes, whereas repression requires Opi1p and occurs when IC are available. In this study, microarray-based expression analysis was performed to assess the global transcriptional response to IC in a wild-type strain and in the opi1delta, ino2delta, and ino4delta null mutant strains. Fifty genes were either activated or repressed by IC in the wild-type strain, including three already known IC-repressed genes. We demonstrated that the IC response was not limited to genes involved in membrane biogenesis, but encompassed various metabolic pathways such as biotin synthesis, one-carbon compound metabolism, nitrogen-containing compound transport and degradation, cell wall organization and biogenesis, and acetyl-CoA metabolism. The expression of a large number of IC-regulated genes did not change in the opi1delta, ino2delta, and ino4delta strains, thus implicating new regulatory elements in the IC response. Our studies revealed that Opi1p, Ino2p, and Ino4p have dual regulatory activities, acting in both positive and negative transcriptional regulation of a large number of genes, most of which are not regulated by IC and only a subset of which is involved in membrane biogenesis. These data provide the first global response profile of yeast to IC and reveal novel regulatory mechanisms by these precursors.