The in vivo activation of lymphokine-producing cells was analyzed in the first 7 days of an acute graft-versus-host reaction (GVHR) induced by injection of C57BL/6 spleen cells into irradiated DBA/2 mice. Although the GVHR was accompanied by a 1000-fold increase in serum IL-6 titers, circulating levels of other lymphokines were low (IL-3, IFN-gamma, and GM-CSF) or undetectable (IL-2 and IL-4). Spleen and lymph node cells from these mice nevertheless produced elevated levels of IL-3, IFN-gamma, GM-CSF, and IL-6 when cultured for 24 h without stimulation; culture with anti-CD3 antibody further increased IL-2, IL-3, IL-4, IFN-gamma, and GM-CSF production by at least 20-fold. Both constitutive and anti-CD3-induced synthesis of all the lymphokines was mediated by CD3+ cells. Messenger RNA analyses revealed the presence of IL-6, IFN-gamma, and GM-CSF transcripts in freshly harvested GVHR spleen cells and increased expression of IL-2, IL-3, IL-4, IFN-gamma, and GM-CSF mRNAs following anti-CD3 stimulation in vitro. In vivo, however, IL-3 mRNA was barely detectable even following cDNA amplification by polymerase chain reaction. In vivo restimulation of day 5 GVHR mice by injection of concanavalin A enhanced expression of IL-3, IL-6, IFN-gamma, and GM-CSF mRNAs and markedly increased serum titers of the corresponding lymphokines, which peaked 6-12 h after injection at levels at least 10- to 100-fold higher than in concanavalin A-treated normal mice.2+ for high-level synthesis of all these lymphokines.