Interferons (IFNs) are potent, pleiotropic cytokines, and therefore it is likely that the cell has mechanisms to modulate IFN activity in response to excessive or prolonged IFN exposure. To investigate this question, Jurkat T cells were exposed to IFN-beta1a in vitro. The effect of dose and frequency of IFN treatment on receptor expression, the signal transduction pathway, and biologic activity was examined. Results demonstrate that at even modest doses of IFN (60 IU/ml), cell surface expression of the IFN receptor subunit, IFNAR-1, decreased significantly, and the cells were unresponsive to further IFN treatment. More interestingly, after an initial treatment with very low concentrations of IFN (<10 IU/ml), even when receptor levels remained normal and phosphorylation of signaling molecules occurred, cells were still refractory to further IFN treatment. After withdrawal of IFN, full cellular responsiveness was a progressive but surprisingly slow process. Cells retreated 2 days or 4 days after the initial IFN treatment were still refractory to even high doses (500 IU/ml) of IFN. Cells retreated 1 week after the initial IFN treatment were fully responsive. High levels of Stat1 and Stat2 correlated with the block in transcriptional activation of IFN-dependent genes and may be a mechanism by which cells can downmodulate an IFN response. Similar results were obtained when fresh peripheral blood mononuclear cells (PBMC) were treated with IFN and expression of the endogenous IFN-dependent gene, MxA, was examined. Cell surface levels of IFNAR-1 decreased and Stat1 levels increased after IFN-beta treatment, and retreatment with IFN resulted in an attenuated induction of Mx protein expression. In the context of using IFNs as therapeutic agents in the treatment of human disease, our data suggest that increasing the amount or frequency of IFN administration may not yield desired biologic effects. Thus, issues concerning the dosage and the frequency of IFN-beta administration deserve careful consideration.