Ionizing radiation shares with cytokines, such as TNF-alpha, an ability to generate free radicals in cells and activate downstream proinflammatory responses through NF-kappaB-dependent signal transduction pathways. Support for the role of free radicals in triggering such responses comes from the use of free radical scavengers like N-acetyl-L-cysteine (NAC). The nature of the link between free radical generation and NF-kappaB activation is, however, unclear. In this study, we explore the possibility that scavenging of free radicals by NAC might not be the mechanism by which it inhibits NF-kappaB activation, but rather that NAC acts through inhibition of proteasome function. The effect of NAC on the chymotryptic function of the 26s and 20s proteasome complex was measured in extracts from EVC 304 bladder carcinoma cells by assessing degradation of fluorogenic substrates. NAC inhibited 26s but not 20s proteasome activity, suggesting that it interferes with 19s regulatory subunit function. NAC blocked radiation-induced NF-kappaB activity in ECV 304 cells and RAW 264.7 macrophages, as measured by a gel shift assay, at doses that inhibited proteasome activity. This provides a possible mechanism whereby NAC could block NF-kappaB activation and affect the expression of other molecules that are dependent on the ubiquitin/proteasome system for their degradation, other than by scavenging free radicals.