Abstract: In the present study we have used DNA microarray and quantitative real-time analysis to examine the transcriptional changes occurring in response to cellular depletion of the yeast acyl-CoA binding protein, Acb1p. Depletion of Acb1p resulted in differential expression of genes encoding for proteins involved in fatty acid and phospholipid synthesis (e.g. FAS1, FAS2, ACC1, OLE1, INO1 and OPI3), in glycolysis and glycerol metabolism (e.g. GPD1 and TDH1), in ion transport/uptake (e.g. ITR1 and HNM1), and in stress response (e.g. HSP12, DDR2 and CTT1). Here, we show that transcription of the INO1 gene, encoding the inositol-3-phosphate synthase, cannot be fully repressed by inositol and choline and that UAS INO1-driven transcription is enhanced in Acb1p depleted cells. In addition the reduced inositol-mediated repression of INO1 transcription observed upon Acb1p depletion appears to be independent of the transcriptional repressor Opi1p. We also demonstrate that INO1 and OPI3 expression can be normalized in Acb1p depleted cells by supplementation of high concentrations of exogenous fatty acids or by overexpressing FAS1 or ACC1. Together, these findings reveal an Acb1p-dependent connection between fatty acid metabolism and transcriptional regulation of phospholipid biosynthesis in yeast. Finally, expression of an Acb1p-mutant unable to bind acyl-CoA esters could not normalize the transcriptional changes caused by Acb1p depletion. This strongly implies that it is either the Acb1p/acyl-CoA ester complex directly or its ability to donate acyl-CoA esters to utilizing systems, which modulates gene expression.