The Cyp 2d-9 gene encodes the male-specific steroid 16α-hydroxylase in mouse liver and shares a conserved regulatory element (2 100 TTCCGGGC 2 93) with another male-specific Slp promoter. As shown with the Slp promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad. Sci. USA 92: 1302–1306, 1995), the male-preferential demethylation also occurs at CpG/297 in the Cyp 2d-9 promoter. The transcription factor which specifically binds to the demethylated element has been purified. The peptide sequences reveal that the factor consists of GABPα and GABPβ1 with Ets and Notch motifs, respectively. Both DNase I footprinting and gel shift assays indicate that the bacterially expressed glutathione S-transferase–GABP fusion proteins bind to the regulatory element only when CpG/297 is demethylated. Moreover, Cyp 2d-9 promoter is trans-activated by coexpression of GABP proteins in HepG2 cells. Given the additional results that CpG/250 of the female-specific steroid 15α-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.