Regulation of I kappaB alpha during activation was examined using EGFP. Single cell analysis showed that both localisation- and cytokine-induced degradation of I kappaB alpha are dependent on expression levels. Cells expressing higher levels of the inhibitor demonstrated an increase in nuclear I kappaB alpha EGFP with a pronounced enhancement in the nuclear/cytoplasmic ratio. Enhancing the levels of the endogenous I kappaB alpha by relA transfection caused significant reduction in IL-1-mediated degradation of the fusion protein. Similarly, I kappaB alpha EGFP-transfected cells showed an inverse correlation between the level of the fusion protein and IL-1-mediated degradation. Comparing absolute levels demonstrated a biphasic response, with reduction in cells expressing over 15-fold that of endogenous levels. Further experiments using Western analysis showed a positive correlation between both phosphorylation and ubiquitination of I kappaB alpha EGFP, and the level the inhibitor. In contrast, and in agreement with the single cell analysis, while IL-1 stimulation caused the expected degradation at lower levels of the fusion protein, breakdown of I kappaB alpha EGFP was totally inhibited at the higher transfection levels. The data show that turnover of I kappaB alpha is saturable and suggest that limitation of the pathway by enhanced inhibitor expression is regulated through a post phosphorylation/ubiquitination event, at the level of degradation.