Loss of deoxyribonuclease I (Dnase1) function is associated with systemic lupus erythematosus (SLE) in humans and mice; however, no coding mutations in Dnase1 are found in polygenic murine models. Instead, both MRL-lpr strains and NZB/W F1 hybrids are homozygous for T89I missense in the macrophage-DNASE, desoxyribonuclease I-like 3 (Dnase1l3). By in vitro expression studies, this substitution decreases this enzyme's nuclease activity against free DNA by only approximately twofold; however, the mutation has a greater effect on the capacity of media conditioned with Dnase1l3 to confer a barrier to liposomal gene transfection to HeLa cells. The 89I substitution decreases the Dnase1l3 barrier function in vitro by eightfold (P < 0·01). In splenocytes and BM-derived macrophages of SLE mice, while cellular Dnase1l3 levels are induced relative to C57BL/6 (control) mice, levels of FD-nuclease activity are similar. Finally, media conditioned by MRL and NZB/W F1 macrophages, relative to control, contains a weak interferon-gamma (IFN-γ) inducible Dnase1l3-associated barrier to transfection. This barrier function is hypothesized to reflect the inability of SLE mice to degrade membrane-enveloped DNA-associated antigens, such as apoptotic bodies, which are predicted to stimulate the characteristic autoimmunity of SLE. Our results for these two generally independent models strongly suggest that Dnase1l3 deficiency increases the susceptibility of these mice to polygenic SLE.