We have shown differences in regulation of cytokine mRNA expression in human tonsil, spleen, and peripheral blood. After PHA stimulation of peripheral blood, IL-2, IL-4, and IL-6 mRNA were expressed with similar kinetics: peak expression occurred after four hours and subsequently declined over 48 hr. In PHA-stimulated splenic and tonsilar MNC, IL-2 and IL-6 mRNA were expressed later, with peak expression occurring after eight hours ofstimulation and no mRNA detectable after 40 hr of stimulation. The intensity of the IL-6 mRNA signal and the amount of IL-6 secreted was much greater in MNC from peripheral blood than in spleen and tonsil and correlated with the percentage of monocytes in MNC from each tissue. IL-4 mRNA expression differed in all three tissues: PHA-stimulated tonsillar MNC expressed IL-4 mRNA with a major peak at eight hours and a minor peak at 24 hr after stimulation. The kinetics of mRNA were not due to effects of mixed cell populations. The same bimodal peak was observed in purified tonsillar T lymphocytes, and the minor 24-hr peak was dependent on IL-4 mRNA synthesis by cells expressing high amounts of VLA-β1 antigen (CDw29) on their surface and which lacked the CD45 epitope recognized by the mAb 2H4. Splenic MNC did not express IL-4 mRNA when stimulated with a number of mitogens and only exhibited IL-4 mRNA expression when stimulated with the combination of PHA and PMA or anti-CD3. Thus, IL-4 mRNA has markedly different kinetics and intensity of expression in spleen, peripheral blood, and tonsil. These differences in cytokine synthesis which vary with anatomic site may be important in understanding the regulation of immune responses in regional lymphoid tissues.