Autoantibodies specific for the Sm ribonucleoprotein are spontaneously produced in patients with SLE and in mice of the MRL mouse strains. We have previously reported the characterization of the clonality and V region gene use of 41 MRL/Mp-lpr/lpr (MRL/lpr)-derived B cell hybridomas selected for Sm binding. In this report, we show that many of the expressed V genes of these hybridomas are also expressed by anti-DNA hybridomas of MRL/lpr mice. Moreover, the anti-Sm hybridomas from nine clonal groups produce antibodies that bind ssDNA, and those of five clones produce antibodies that also bind dsDNA. Sm/DNA-specific hybridomas, but not Sm-only-specific hybridomas, have a higher than expected content of arginine residues in CDR3 of the H chain, similar to MRL/lpr hybridomas selected on the basis of DNA binding. One clone displays intraclonal differences in DNA binding, inasmuch as the most extensively mutated members produce antibodies that are able to bind dsDNA and have a higher affinity for ssDNA than the least mutated members of this clone. Thus, DNA appears to be a selecting Ag in this response. These data indicate an overlap in the anti-Sm and anti-DNA autoimmune responses in MRL mice that may have implications for the activation of anti-Sm B cells, and for defining the spectrum of Ag targeted in SLE.