Potentially, autoimmune diseases develop from a combination of multiple genes with allelic polymorphisms. An MRL/Mp‐Fas^lpr/lpr (MRL/lpr) strain of mice develops autoimmune diseases, including lupus nephritis, but another lpr strain, C3H/HeJ‐Fas^lpr/lpr (C3H/lpr) does not. This indicates that MRL polymorphic genes are involved in the development of the diseases. By quantitative trait loci (QTL) analysis using 527 of the (MRL/lpr × C3H/lpr)F₂ mice, we identified a novel locus for susceptibility to lupus nephritis at map position D5Mit115 on chromosome 5, the same alias of the osteopontin (Opn) gene (LOD score =4.0), susceptible in the MRL allele. In functional analyses of the MRL and C3H Opn alleles using synthetic osteopontin (OPN) made with a new method “cell‐free system” with wheat germ ribosomes, the MRL‐OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF‐α, IL‐1β and IFN‐γ in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis.