Id2, a negative regulator of basic helix-loop-helix transcription factors, is involved in regulating cell differentiation and proliferation. To obtain insight into the role of Id2 in cell cycle control, we investigated the mechanisms underlying the immediate early response of Id2 expression to serum stimulation in NIH3T3 cells. Luciferase reporter analysis with deletion and point mutants demonstrated the serum response element of Id2 (Id2-SRE) to be a consensus binding site for RFX1 (regulatory factor for X-box 1) present 3.0 kb upstream of the transcription initiation site of Id2. Gel shift and chromatin immunoprecipitation assays confirmed the binding of RFX1 to Id2-SRE in vitro and in vivo, respectively. In both assays, RFX1 binding was observed not only in serum-stimulated cells, but also in serum-starved cells. Knockdown of RFX1 by RNA interference disturbed the immediate early response of Id2 expression in cells and abrogated the Id2-SRE-mediated induction of luciferase activity by serum. These alterations were rescued by the introduction of RNA interference-resistant RFX1 into cells. On the other hand, in the Id2-SRE-mediated reporter assay, RFX1 with an N-terminal deletion abrogated the serum response, whereas RFX1 with a C-terminal deletion enhanced the reporter activity in serum-starved cells. Furthermore, HDAC1 was recruited to Id2-SRE in serum-starved cells. These results demonstrate that RFX1 mediates the immediate early response of the Id2 gene by serum stimulation and suggest that the function of RFX1 is regulated intramolecularly in its suppression in growth-arrested cells. Our results unveil a novel transcriptional control of immediate early gene expression.