Lipopolysaccharide (LPS) is a B cell mitogen which can stimulate murine primary B cells to proliferate and carry out immunoglobulin heavy chain class switch recombination. LPS can also function as an endotoxin, which may cause DNA damage and apoptosis in certain types of cells. We have previously reported that LPS-activated primary murine B cells contain nuclear loci that stain brightly with anti-Rad51 antibodies (Li et al. (1996) Proc. Natl. Acad. Sci. USA 93, 10222-10227). We have now analyzed Rad51 nuclear loci induced in both primary and immortalized B cells by treatment with the DNA damaging agent, methyl methanesulfonate (MMS). We have found that, in LPS-cultured primary B cells, MMS treatment increases the fraction of cells containing Rad51 loci and induces formation of a very high number of loci per cell. The loci induced by MMS treatment are small, punctate, and numerous; in contrast, the loci induced by LPS activation are large, brightly staining, and relatively few in number. In LPS-cultured primary B cells, Rad51 relocalizes during the cell cycle, and large, brightly staining nuclear loci are present in only restricted stages of the cell cycle. Rad51 foci similar to those present in LPS-activated primary B cells are also observed in immortalized B cell lines cultured in the absence of LPS. These loci are unaltered in number or appearance by culture with LPS, but treatment of immortalized B cell lines with MMS induces loci which are small and punctate in staining, like those induced by MMS in primary B cells. These data show that distinctive Rad51 loci are induced by DNA damaging agents and cell activation and that the response to DNA damage may involve pathways distinct from those associated with B cell activation and switch recombination.