AbstractPrevious studies from this laboratory indicated that the product of the RSF1 gene of S. cerevisiae is present in both nucleus and mitochondria, and they suggested that Rsf1p acts as a transcriptional modulator. To investigate this latter question, we performed transcriptome profiling of an rsf1 mutant strain and its wild‐type parent during a shift from glucose‐based fermentative to glycerol‐based respiratory growth to identify genes whose expression is regulated by Rsf1p. Loss of Rsf1p engendered a decrease in transcript levels from many genes encoding components of the electron transport chain and various other mitochondrially‐localized products. The earlier studies further showed that rsf1 cells exhibit a growth defect on medium containing glycerol, but not ethanol, as sole carbon source. Importantly, transcriptome profiling of the rsf1 mutant during shift from glucose‐ to glycerol‐based medium revealed that the product of this gene plays a major role in both orchestration of the transition to, and maintenance of, efficient growth on glycerol as sole carbon source. An increase in transcript levels from genes encoding products that function in the stress response, and an imbalance between expression of genes encoding glycerol anabolic and catabolic enzymes, was observed in the rsf1 mutant during steady‐state growth on glycerol‐ but not ethanol‐based medium; this suggests the presence of partially separate transcriptional regulatory systems for transition to respiratory growth on each of these two carbon sources. Genes whose expression is affected by loss of Rsf1p, which lacks a known DNA‐binding motif, lack a common DNA sequence motif in their upstream regions. These and other data presented here strongly suggest that the transcriptional effects exerted by Rsf1p are mediated via interaction with other transcription factors. Copyright © 2009 John Wiley & Sons, Ltd.