Murine helper T lymphocyte (HTL) clones reactive to ovalbumin (OVA) were maintained in continuous culture in vitro. Clones were propagated by weekly stimulation in the presence of irradiated splenic filler cells, antigen, and supernatant fluid (SF) containing IL 2. By varying the quantity of these reagents in cultures of HTL cells, the reactivity to antigen of the cloned cells was altered markedly. After stimulation by antigen or SF, HTL clones became profoundly unresponsive to antigenic restimulation. Cells remained unresponsive for 2 to 9 days after stimulation, depending on the culture conditions that were chosen for their maintenance. The addition of SF containing a high concentration of IL 2 prolonged the duration of unresponsiveness by 3 days, and the presence of a non-T splenic filler cell increased the period of unresponsiveness by an additional 4 days. The use of a high concentration of OVA in cell cultures also prolonged the time of unresponsiveness. The results described here demonstrate that the response to antigen of HTL cells is down-regulated after stimulation, and appears to be correlated with exposure to SF that contains IL 2.