Salmonella typhimurium mitogen (STM) is a polyclonal activator of rat B lymphocytes, triggering them to proliferate, but not differentiate, to antibody-secreting cells. When lymphokines in the form of a supernatant from Con A-stimulated splenocytes (CAS) are added to B cell cultures activated by STM, only a small number of cells are driven to differentiate. Only with the addition of a third signal provided by the polyanionic polysaccharide dextran sulfate (DXS) is significant rat B cell differentiation observed. In this study, we have shown that this requirement for DXS is not unique to the STM mitogen. LPS, Staphylococcus aureus Cowan I-fixed cells, and anti-Ig antibody all induced rat B cell proliferation with little differentiation, even in the presence of CAS. DXS was necessary to induce differentiation in all cultures costimulated with mitogen and CAS. The requirement for DXS for optimal B cell differentiation is also observed with other lymphokine preparations such as the supernatants from PMA-stimulated EL-4 cells and PHA-stimulated human T cells. Furthermore, this augmentative effect of DXS in rat B cell differentiation was not confined to polyclonal activation systems. Ag-specific IgG secretion was also increased when DXS was added to Ag and CAS costimulated cultures of B cells harvested from the draining lymph nodes of rats immunized with DNP-keyhole limpet hemocyanin. Within the polyclonal activation system, a method of staged additions of STM, DXS, and CAS to B cell cultures was used to investigate the role of DXS during B cell differentiation. Optimal differentiation occurred only when DXS was present in the B cell cultures in conjunction with CAS. The augmentation in differentiation seen with DXS did not appear to be due to the recruitment of an additional CAS-responsive B cell subset, because cycling, low density B cell blasts showed large increases in IgM secretion with subsequent exposure to DXS and CAS. These studies suggest tha DXS acts as a cofactor to various differentiation factors, augmenting polyclonal and Ag-specific rat B cell differentiation. The relevance of DXS to in vivo immune responses is discussed.