Genetic polymorphism of murine C3 was demonstrated by a combined use of isoelectric focusing (IEF) in thin layer polyacrylamide gel and immunofixation. All 18 inbred strains, which were originally derived from the United States, showed a single C3 band of pI 6.1. In contrast, two strains (NC and NBr) derived from the Japanese fancy mouse and a strain (MoA), which was established from Japanese wild mouse (Mus musculus molossinus), showed a single but more anodal C3 band of pI 6.0. A mixture of plasma from two different strains, one from the Japanese mouse and another from the mouse of American origin, resulted in two discrete C3 bands; neither of these two bands can be distinguished from the C3 band observed in one of the unmixed plasmas. Breeding experiments established that the pI variation of mouse C3 is inherited as an autosomal co-dominant trait controlled by a single locus. Thus, the C3 gene segregated in the classical Mendelian ratio among second generation progeny from a cross between NC and one of the American strains, B10, BALB/c, or CBA. The locus controlling the pI variation of murine C3 was designated as C3-1 locus and its linkage with other genetic markers was studied. The C3-1 locus segregated relative to Albino, Agouti, Brown, Es-1, HC, and Id-1. However, the linkage of the C3-1 locus to two loci, Ce-2 and the S region of the H-2 complex was observed. Both of these two loci are localized to chromosome 17. The distances of the C3-1 locus from Ce-2 and S were estimated as 23 and 11 centimorgans, respectively. These values imply that the C3-1 locus is located outside H-2 of the chromosome 17.