Electrophoretic mobility shift assays (EMSA) were used to define the regions of the INO1 promoter that interact with factors present in extracts prepared from the yeast, Saccharomyces cerevisae. These experiments identified three different types of protein:DNA complexes that assemble with the INO1 promoter. Formation of one type of complex depended on functional alleles of previously described regulatory genes, INO2 and INO4, that encode positive regulatory factors. Formation of the INO2/INO4-dependent complexes was increased when extracts prepared from cells grown under derepressing conditions (i.e. absence of inositol and choline). A second type of complex was dependent on the OPI1 gene, that encodes a negative regulator. Computer-aided sequence analysis of the promoters of several genes encoding phospholipid biosynthetic enzymes revealed a highly conserved nine basepair element (5'-ATGTGAAAT-3'), designated 'nonamer' motif, that is similar to the octamer motif identified in the promoters of mammalian immunoglobulin genes. The nonamer motif was shown to form a specific complex with a factor, designated nonamer binding factor (NBF). Extracts prepared from Schizosaccharomyces pombe formed a nonamer-specific complex.