The fourth component of C (C4) and sex-limited protein (Slp) genes of FM strain mice show more than 95% nucleotide identity both in the coding and the about 2 kb of 5' flanking region, but are distinct in their regulation. Although C4 is a constitutive gene, Slp is an androgen-dependent gene and shows negligible basal level expression. We have previously shown that this difference in basal expression is determined by a region of about 400 bp immediately 5' of the transcriptional start site of the C4 and Slp genes. In this report, we demonstrate a specific binding site for a HepG2 nuclear extract in this region of the C4 gene by gel retardation and DNase I footprinting experiments. Nucleotide sequence of this binding site is very similar to the recognition sequence of well characterized transcription factors, NF.kappa B and H2TF1. Synthetic oligonucleotides representing the binding sites of the H-2Kb class I and IL-6 genes for these factors compete with the C4 promoter for the complex formation with a HepG2 nuclear factor. Due to the nucleotide deletion and substitution, the corresponding region of the Slp gene lacks this sequence motif as well as binding activity to the HepG2 nuclear factor.