Macrophages (mφ) from prediseased mice of all the major murine models of spontaneous autoimmunity have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. We show here that mφ from prediseased mice of the same models of spontaneous autoimmunity share a serum-dependent defect in the activity of Rho, a cytoplasmic G protein and cytoskeletal regulator. Affected strains include those developing lupus (BXSB, LG, MRL/l+, MRL/lpr, NZBWF1) and autoimmune diabetes (nonobese diabetic). No similar defect in Rho activity occurred in seven control strains. In the presence of serum, Rho activity in mφ from all autoimmune-prone strains was reduced to less than 10% of that in control mice. In contrast, under serum-free conditions, Rho activity was ompletely normal in autoimmune-prone mφ. The activities of Ras, another cytoplasmic G protein, and Rac and Cdc42, two additional G protein regulators of the cytoskeleton, were regulated normally in autoimmune-prone strains. Serum-dependent dysregulation of Rho was associated with multiple abnormalities, including increased adhesion to various surfaces, a more spread dendritic morphology, and an altered actin cytoskeletal organization. Our results suggest that mφ from multiple, genetically diverse, autoimmune-prone strains share a mutation or allelic difference affecting signal transduction within a specific Rho-regulatory pathway.