In Saccharomyces cerevisiae, the phospholipid biosynthetic genes are transcriptionally regulated in response to inositol and choline. This regulation requires the transcriptional activator proteins Ino4p and Ino2p, which form a heterodimer that binds to the UASINO element. We have previously shown that the promoters of the INO4 and INO2 genes are among the weakest promoters characterized in yeast. Because little is known about the promoters of weakly expressed yeast genes, we report here the analysis of the constitutive INO4 promoter. Promoter deletion constructs scanning 1,000 bp upstream of theINO4 gene identified a small region (−58 to −46) that is absolutely required for expression. S1 nuclease mapping shows that this region contains the transcription start sites for the INO4gene. An additional element (−114 to −86) modestly enhancesINO4 promoter activity (fivefold). Thus, the region required for INO4 transcription is limited to 68 bp. These studies also found that INO4 gene expression is not autoregulated by Ino2p and Ino4p, despite the presence of a putative UASINO element in the INO4promoter. We further report that the INO4 steady-state transcript levels and Ino4p levels are regulated twofold in response to inositol and choline, suggesting a posttranscriptional mechanism of regulation.