Adherent peritoneal cells, presumably macrophages, from mice of a number of congenic strains when cultured in vitro in medium containing 14C-labeled amino acids were found to synthesize the Ss protein (mouse C4), as revealed by immunoprecipitation with anti-Ss serum from 14C-labeled, 24-hr culture supernatants. Strain B10.WR7, which expresses the Slp allotype of the Ss protein, was shown by the same method also to synthesize the Slp antigen. SDS-solubilization and 2-ME-reduction of the B10.WR7 immunoprecipitates followed by separation on SDS polyacrylamide slab gels and autoradiography revealed three major polypeptide bands in Slp-positive Ss molecules and three major bands in Slp-negative Ss molecules with m.w. comparable to those of the α-, β-, and γ-chains of human C4. The Slp-negative Ss polypeptides of strain B10.WR7 had apparent m.w. of 98,000, 75,000, and 33,500 whereas the Slp-positive Ss molecules of B10.WR7 yielded three polypeptides with apparent m.w. of 105,500, 75,000, and 32,000. Immunoprecipitation analyses of normal, unlabeled B10.WR7 serum or plasma in SDS slab gels followed by staining with Coomassie Blue revealed similar differences in the m.w. of the α- and γ-polypeptides of the Slp-negative versus the Slp-positive Ss proteins. Such analyses of sera and plasmas of mice from a backcross linkage test and from a series of intra-H-2 recombinants showed clearly that the m.w. differences in α- and γ-chains are controlled by the S region of the H-2 complex. In addition, the Slp-negative Ss protein in strains other than B10.WR7 had an Ssα-chain of 103,000 daltons, indicating the existence of still further Ss polymorphism also controlled by the S region. An apparent intracellular precursor polypeptide of the Ss molecule with a m.w. of approximately 185,000 (comparable to the guinea pig Pro-C4 described recently) has also been demonstrated in lysates of cultured peritoneal cells. These results further confirm the structural homologies of the mouse Ss-Slp and human and guinea pig C4 proteins. They also indicate that Ss and Slp-antisera define two similar, but structurally distinct subclasses of mouse C4 that are apparently controlled by two discrete structural genes in the S regions of Slppositive strains. It is postulated that each of these structural genes codes for a single polypeptide precursor for the native Slp-positive and Slp-negative Ss molecules, and that the observed differences in the m.w. of the α- and γ-chains of these two Ss subclasses reflect a mutational shift in the site of the proteolytic cleavage step that releases the chains.