The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins that exhibit very disparate patterns of gene expression: all mice constitutively express C4, whereas only adult male mice from a limited number of standard inbred strains express Slp. Several exceptional strains exhibit constitutive (C4-like) Slp expression, a phenotype that correlates with multiple copies of the Slp gene. To determine the molecular basis for constitutive Slp expression we have isolated genomic clones and compared the sequences of 1.5 kb of 5' flanking DNA from 1 C4 gene and three different Slp genes from the Slp-constitutive strain B10.WR. These sequence comparisons demonstrate C4-like regulatory sequences adjacent to two of the Slp genes. By analysis of cDNA clones isolated from a B10.WR liver library we demonstrate that the constitutive Slp phenotype is due primarily to expression of one of these C4/Slp hybrid genes. It appears likely that Slp gene duplication in strain B10.WR came about via homologous unequal crossover events between C4 and Slp genes; this would accommodate both the gene sequence data and the pattern of C4-like Slp expression in mouse strain B10.WR.