The precise molecular characteristics and the mode of action of the T cell derived lymphokines which augment antibody production in vitro remain uncertain. The use of ill-defined culture supernatants to dissect the cellular interactions in vitro involved in antibody production can lead to ambiguous results as the factors may act either on a contaminating non-B-lymphoid population or directly on the B lymphocyte. We report herein the development of a system for measuring in vitro primary antibody responses by murine spleen cells in which endogenous lymphokine production has been minimized by the in vivo administration of cytotoxic antibodies to deplete T lymphocytes and the addition of the glucocorticosteroid, dexamethasone, throughout the culture period. Using such an assay, a lymphokine activity was detected which was capable of augmenting the plaque forming cell response. This lymphokine was present in culture supernatant derived from the lectin activation of the T cell lymphoma, LBRM-33 and was distinct from other known B cell activators, notably IL-2 and IFN gamma. Biochemical purification of this activity indicated that it might be identical to granulocyte-macrophage colony stimulating factor (GM-CSF). The use of recombinant-derived GM-CSF protein unambiguously showed the role of this lymphokine in antibody production. These experiments demonstrated for the first time, the involvement of a hematopoietic factor in antigen-specific immune responses. Moreover, these results demonstrated an important regulatory circuit in the generation of antibody producing B cells in which GM-CSF, derived from activated T cells, stimulates macrophage function.