Although DNA is generally considered to be a poor immunogen, recent evidence suggests that DNA from various species differ in their immunologic activity and that bacterial DNA, unlike mammalian DNA, can induce significant antibody responses in mice. To explore further the immunologic activities of bacterial DNA, its ability to stimulate in vitro proliferation of murine lymphocytes was tested. The stimulation of lymphocytes with highly purified ssDNA from Escherichia coli resulted in a dose-dependent response that was maximal at 48 h. Several lines of evidence indicate that DNA, rather than endotoxin contamination, induced this response: 1) LPS at doses equivalent to those detected in the DNA preparation caused significantly less proliferation than the DNA; 2) the response to DNA was insensitive to polymyxin B; 3) pretreatment of DNA with DNase completely abrogated the response; and 4) DNA induced the proliferation of cells from endotoxin-resistant C3H/HeJ mice. Furthermore, although DNA from three different bacterial species induced proliferation, mammalian DNA from three species were nonmitogenic. Depletion of T cells from lymphocytes did not reduce proliferation, suggesting that bacterial DNA directly triggered B cell proliferation. These studies provide further evidence that DNA are not uniform in their immunologic activities likely because of their content of nonconserved structural determinants.