We have identified and characterized a specific nuclease activity to be tightly associated with proteasomes. Using tobacco mosaic virus RNA (TMV-RNA) as substrate to analyze and quantify the cleavage reaction, we supply several lines of evidence that this nuclease activity is an integral part of proteasomes. Thus, RNase activity was coincident with the elution profiles of proteasomes at each stage of purification. Proteasomal nuclease activity was resistant to strong dissociation conditions using 480 mM KCl, 0.5% sodium lauroylsarcosinate, and 6 M urea. This nuclease activity remained associated with an urea-resistant subcomplex of the proteasome comprising a specific set of proteins. Finally the digestion of TMV-RNA led to a well defined pattern of RNA fragments while 5 S ribosomal RNA and globin mRNA were not degraded. These results provide further evidence that proteasomes are able to discriminate between different RNAs, and the possible involvement of proteasomes in translation control is discussed.