Studies of cellular events associated with antigen-induced triggering and differentiation of B cells would be greatly facilitated by the availability of homogeneous cell lines of antigen-specific lymphocytes that can be maintained in long-term culture. By combining the techniques of enrichment of lymphocytes for antigen-specific cells, cloning in soft agar, and long-term propagation of B cells we have been able to isolate, propagate, and maintain two lines of dinitrophenyl (DNP) -specific B lymphocytes. These cell lines are B lymphocytes that have 70% and greater than 80% DNP-specific rosette-forming cells, respectively. Both cell lines secrete small amounts of antibody spontaneously but can be stimulated by antigen in vitro in the presence of either supernatants from phytohemagglutinin-stimulated spleen cells or irradiated normal filler cells. Thus far these lines have been maintained in vitro for greater than 9 mo. They will be useful in studying factors associated with B cell response.