Lymphocyte blastogenesis assays and immunoblotting were used to investigate and compare murine B-cell responses to preparations of extracellular membrane blebs (BAg) and spirochetes (Ag) of Borrelia burgdorferi. Immunoblotting BAg, Ag, and medium control preparations with serum from naive and infected C57BL/10 mice revealed that BAg and Ag had similar specific reactivity profiles except that major antigens of 83, 60, and 41 kDa were detected in Ag but not in BAg. It was determined that 1 microgram (dry weight) of Ag contained 0.0051 and 0.0063 microgram of outer surface proteins A (OspA) and OspB, respectively, whereas 1 microgram (dry weight) of BAg contained 0.0024 microgram of OspA and 0.0015 microgram of OspB. Both BAg and Ag caused blastogenesis in cultures of spleen cells from both groups of mice, but BAg-stimulated lymphocytes exhibited significantly greater (P < or = 0.05) blastogenesis after 2 or 6 days of culture than did lymphocytes stimulated by Ag or medium control. Flow cytometry and antibody capture enzyme-linked immunosorbent assays identified responding lymphocytes as B cells which secreted polyclonal immunoglobulin M (IgM) but not IgG or IgA. Treatment of BAg and lipopolysaccharide controls with polymyxin B resulted in as much as 20.7 and 54.3% mean decreases in blastogenesis, respectively. Fractionation of BAg or Ag by ultracentrifugation before culture with spleen cells from naive mice indicated that B-cell blastogenesis was probably associated with spirochetal membranes. The results of this study demonstrate that specific humoral responses are directed towards extracellular membrane blebs which lack the 83-, 60-, and 41-kDa antigens of intact spirochetes and that blebs also possess significant nonspecific mitogenic activity for murine B cells. This activity was not due entirely to typical lipopolysaccharide or OspA and OspB lipoproteins.