The translocation of the transcription factor NF-kappaB into the nucleus plays a critical role in many physiological events. In unstimulated cells, NF-kappaB is sequestered in the cytosol, bound to its inhibitor IkappaB. Activation primarily occurs via the IkappaB kinase (IKK) complex which phosphorylates IkappaBalpha at serines 32 and 36, creating a recognition site for IkappaB ubiquitination which then targets IkappaB for degradation. Often it is useful to measure IKK activity to assess upstream signaling events leading to NF-kappaB activation. Current methods of assessing IKK activity are limited to IKK isoforms which are recognized by available IKK antibodies. Here, we describe a procedure to qualitatively assess the overall IKK activity in a cell lysate which can be used on any IKK isoform capable of phosphorylating human IkappaBalpha. This nonradioactive assay is based on measurement of the ability of the cell lysate to phosphorylate GST-IkappaBalpha, as measured by Western blotting, using an anti-phospho-IkappaBalpha antibody. We have used this assay to observe the kinetics of TCR-mediated activation of IKK as compared to PMA/ionomycin in primary rat T cells. PMA/ionomycin induces maximal IKK activity within 1 min of stimulation and this activity remains elevated for over 20 min. In comparison, TCR ligation induces maximal IKK activity after 5 min of stimulation and this activity rapidly diminishes to background levels. These data indicate that different stimuli can activate and inactivate IKK with different kinetics and suggest that TCR-mediated activation of IKK is closely linked to the rapid phosphorylation and dephosphorylation, respectively, of TCR-associated kinases.