An in vitro culture system for the induction of an antipolysaccharide response was used to study the cellular interactions which determine the magnitude and nature of this B-lymphocyte response. Healthy adult volunteers were vaccinated with the Haemophilus influenzae type b polysaccharide (PRP)-tetanus toxoid (TT) conjugate vaccine. Optimal in vitro anti-PRP and anti-TT antibody responses were obtained when B cells were cultured with equal amounts of T cells. The in vitro response is antigen dependent and antigen specific. Culturing with PRP mixed with TT in the presence of T cells induces the highest number of anti-PRP antibody-secreting cells (ASC) (128.4 x// 15.9 [geometric mean x// standard deviation] immunoglobulin M [IgM] anti-PRP ASC/10(6) cells; 9.3 x// 7.6 IgG anti-PRP ASC/10(6) cells). Culturing without T cells induced no anti-PRP ASC; culturing with only PRP, in the presence of T cells, yielded low numbers of anti-PRP ASC (3.7 x// 5.2 IgM anti-PRP ASC/10(6) cells and 1.2 x// 2.2 IgG anti-PRP ASC/10(6) cells). Transwell studies showed that the requirements for the antibody response against the polysaccharide are different from those of an antiprotein response. Cytokines formed as a consequence of contact between protein-specific B and T cells were on their own not sufficient to activate TT-specific B cells (8.4 x// 1.4 anti-TT ASC/10(6) cells); direct contact between T and B cells appeared to be an absolute requirement. However, physical contact between B and T cells in one compartment of the Transwell system resulted in the release of soluble factors able to stimulate B cells in the other compartment to secrete antipolysaccharide antibodies (164 x// 1.6 anti-PRP ASC/10(6) cells).