The Ino2 and Ino4 proteins belong to the basic helix-loop-helix (bHLH) family of transcription factors. These two proteins are required for the induction of phospholipid biosynthetic gene expression in response to inositol deprivation. The Ino2/Ino4 heterodimer binds a 10 bp region (UASINO) present in the promoters of the phospholipid biosynthetic genes. Recent studies show that expression of an INO2-cat reporter gene is autoregulated by the INO2 and INO4 genes through a UASINO element present in the INO2 promoter. Expression of an INO4-cat reporter gene is unresponsive to inositol and requires the INO4 gene but not the INO2 gene. These observations, coupled with the inability of the Ino4 protein to activate transcription on its own, suggest that Ino4 may partner with multiple proteins. Thus, the INO4 gene product may form multiple dimer combinations which may control expression of different sets of target genes. While multiple pairings for bHLH proteins is common in mammalian cells, this is the first case in yeast. We have initiated experiments to investigate the cis- and trans-acting regulatory elements required for INO4 expression. We have made deletion constructs of the INO4 promoter and fused them upstream of the cat gene. These experiments have identified two regions of the INO4 promoter that are required for expression. Mobility shift assays are being conducted to examine the protein complexes that assemble on the INO4 promoter.