Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-kappaB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-kappaB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IkappaB-alpha degradation. Here we report the construction of p65/NF-kappaB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,120) --> Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-kappaB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Although a slight inhibition of IkappaB degradation was detected for all SLs, the amount of remaining IkappaB was too low to explain the observed NF-kappaB inhibition.