The INO2 gene of Saccharomyces cerevisiae is required for derepression of the phospholipid biosynthetic genes in response to inositol depletion. Conversely, the OPI1 gene is required for repression in response to inositol supplementation. Results of an in vitro assay have led to a model in which Opi1p interacts with Ino2p. However, there is no in vivo evidence to support this model. Additionally, most of the previously isolated ino2 mutants offer little insight into this model. Here, we report the isolation of a new class of dominant mutations in the INO2 gene, which yield constitutive expression of a target gene (i.e. an Opi(-) mutant phenotype). Two mutations reside in a region of the Ino2p required for interaction with Opi1p in vitro. Three other mutations are at the amino-terminus in a transcriptional activation domain.