Using a culture of cardiomyocytes it has been shown, that a well-known inhibitor of autophagy, N-3-methyladenine causes a 1.4 fold increase (p = 0.023) of the chymotrypsin-like activity, a 1.5 fold increase (p = 0.09) of the peptidyl-glutamyl peptide-hydrolyzing activity and 1.5 fold decrease (p = 0.07) of the trypsin-like activity of the proteasome. N-3-methyladenine in a dose-dependent manner inhibits chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the purified 20S proteasome, but activates it trypsin-like activity. Chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the 26S proteasome from proteasome fraction II did change in the same way, as in the case of 20S proteasome, but trypsin-like activity decreased. Using the above method of determining ribonuclease activity, we have shown, that N-3-methyladenine and clasto-lactacystin b-lactone inhibit the RNase activity of the proteasome. Specific proteasome inhibitor exhibits more powerful action, almost completely preventing RNA of actin and myosin from degradation. These data show a multitarget action of N-3-methyladenine, resulting in changes of peptidase and ribonuclease activity of the proteasome.