K48R ubiquitin (K48R-Ub) is an analogue of native ubiquitin that does not form polyubiquitin chain conjugates. Targeted delivery of this recombinant mutant ubiquitin to human macrophages results in an intracellular increase in the ubiquitin analogue. IkappaBalpha polyubiquitination and degradation were significantly inhibited in K48R-Ub targeted macrophages upon stimulation with lipopolysaccharide. The ability to reduce IkappaBalpha degradation was also associated with a reduced production of TNF-alpha, the gene of which is under NF-kappaB control. At a concentration of 0.1 microM, dexamethasone was less effective than K48R-Ub in preventing IkappaBalpha depletion and TNF-alpha release. These data suggest that ubiquitin analogues are potent suppressors of TNF-alpha release in macrophages.