The requirements for different activation signals in the generation of plaque-forming cell (PFC) responses by positively selected B (surface immunoglobulin-positive) cells were analyzed in low-density cultures to minimize the possible effects of contaminating T cells. Using this system, it is demonstrated that not only in T helper cell (TH)-dependent but also in lipopolysaccharide (LPS)-dependent (i.e., so-called T-independent) PFC responses, the resting B cells have to receive at least three different signals: (a) a major histocompatibility complex (MHC)-specific TH signal that can be bypassed by LPS, (b) an antigen signal, and (c) a second TH signal medicated by MHC- and antigen-unspecific helper factor(s) for B cell responses (BHF) that cannot by bypassed by LPS. Specifically, contact of surface immunoglobulin-positive cells with cloned allo-I-A-specific TH or LPS induced a polyclonal PFC response without significant proliferation, whereas contact with BHF alone (obtained as supernatants from different cloned TH, EL-4 thymoma cells, or secondary mixed leukocyte culture cells) had no effect. Only when LPS, antigen, and BHF, or, alternatively, allo-TH (producing themselves BHF) and antigen were present did clonally expanded PFC responses occur. Thus, the data indicate that both an LPS (or specific TH) signal and an antigen signal are required to render the B cells responsive to BHF. BHF seems to act essentially as a nonspecific growth factor, whereas differentiation into antibody-secreting cells appears to be a preprogrammed consequence of B cell activation by an LPS or specific TH signal.