Mouse Slp, a duplicate of the fourth complement component (C4) gene, exhibits EDTA-independent complement activity with a hepatic expression that is male specific. To provide an underlying mechanism for the male-specific expression, we have analyzed the promoter activity of the various 5'-flanking sequences and CpG demethylation of the Slp gene. Transient transfections using HepG2 cells indicate that the element TTCCGGGC (nt -124 to -117) regulates the promoter activity. Moreover, CpG at position -121 of this regulatory element is demethylated to a much higher degree in males than in females. This sexually dimorphic DNA demethylation is consistent with the male-specific expression of the Slp gene in DBA/2 males. The regulatory element binds to the different TTCCGGGC-specific nuclear proteins depending on the methylation of the CpG site. In contrast, the corresponding CpG at position -119 of the C4 gene, which is expressed in both males and females, is demethylated at equal and high levels in both sexes. We therefore propose that the DNA demethylation and methylation-sensitive transcription factors may be a part of the regulatory mechanism for the male-specific expression of the Slp gene.