To investigate the potential of DNA to elicit immune responses, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell depleted human peripheral blood B cells. Among 34 ODNs tested, three specific phosphorothioates (27bp antisense to rev gene of HIV, 21 bp antisense to HSV, and 20bp randomer) induced B cell proliferation and Ig production at low concentrations (1-5 ng/ml). IL2 augmented both proliferation and production of IgM, IgG and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, intact T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active ODNs, more than 90% of B cells expressed CD25 and CD86. In addition, B cells stimulated with ODNs expressed all 6 of the major immune-globulin VH gene families. These results indicate that the human B cell response to ODNs is polyclonal. Active ODNs coupled to sepharose beads stimulated B cells as effectively as the free ODNs, indicating that stimulation resulted from engagement of surface receptors. These data indicate that specific ODNs can directly induce polyclonal activation of human B celts in a T cell-independent manner by engaging as yet unknown B cell surface receptors.