This paper describes a novel primary in vivo/in vitro culture system which allows analysis of the effect of IL-4 added to culture 1 day after immunization on the production of IFN-gamma. Mice are immunized epicutaneously with picryl chloride (TNP) and draining lymph node cells were harvested 1 day later. These cells (1 day lymph node cells), when cultured in vitro for 3 days in the presence of IL-2, either continuously or as a pulse, give an IFN-gamma response on reexposure to antigen 3 days later. This production of IFN-gamma is both antigen-specific and genetically (MHC)-restricted and is due to both CD8+ and CD4+ T cells. However, if 1 day lymph node cells are cultured with both IL-2 and IL-4, no IFN-gamma is produced on subsequent reexposure to antigen, but the cells acquire the ability to produce IL-4 and IL-10. Moreover, cells pulsed with IL-4 blocked IFN-gamma production when cocultured with cells pulsed with IL-2. IL-4 only exerted its inhibitory activity on IFN-gamma production when added on the first or second day of culture and had no effect at later times. Finally, the inhibitory activity of IL-4 on IFN-gamma production may depend on the production of IL-10, induced by the IL-4, as the inhibitory effect of IL-4 is reversed by mAb against IL-10.