In Saccharomyces cerevisiae, the expression of several genes encoding enzymes involved in lipid metabolism is regulated by inositol and choline. The transcriptional heterodimeric complex composed of the gene products of INO2 and INO4 binds to a conserved cis-acting upstream activating sequence designated as the inositol-choline responsive element (ICRE), and activates the expression of these genes. In the presence of inositol and choline, the expression of these genes is down-regulated and a functional OPI1 gene product is necessary for this repression. The promoter region of OPI1 contains one copy of ICRE, and here we analyzed the involvement of ICRE in the inositol-choline-mediated gene regulation of OPI1. Deletion analysis of the OPI1 promoter region, disruption of ICRE in it, and its activity in ino2- and ino4-disrupted strains showed that ICRE is essential for the expression of OPI1, and that the expression of OPI1 is dependent on the INO2 and INO4 gene products. Disruption of OPI1 resulted in the derepressed expression of OPI1 itself and no response to inositol-choline, showing that OPI1 is regulated in the same manner as the phospholipid biosynthetic genes. These results revealed the regulatory circuit of the expression of the positive regulatory gene INO2 and the negative regulatory gene OPI1.