The fate of the proteasome-generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide-degrading and -binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin 2572264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo-peptidase motif.Altogether, the results presented here suggest that Zn 21 at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal-generated oligopeptides in the cytosol.Keywords: thimet oligopeptidase; ATP; autophosphorylation; chaperone.In eucaryotic cells most of the cytosolic peptides generated by the proteasome undergoes complete degradation by different classes of peptidases [1]. This process of intracellular protein degradation is part of the homeostatic mechanism, essential for the structure and function of the cells (reviewed in [2]) However, few peptides, which accomplish important biological tasks, are prevented from degradation by poorly understood mechanisms. This is the case of the MHC class I peptide epitopes which are presented at the cell surface in complex with the MHC class I molecule [3]. Our previous investigation on the susceptibility of a broad spectrum of randomly chosen MHC class I peptide epitopes to the thiol-activated metallo-oligopeptidase (TOP) indicated that in vitro few of them were degraded while the majority was resistant. Similar results were obtained using crude macrophage cytosol. Surprisingly, the majority of the epitopes, which were resistant to TOP degradation, displayed high affinity to the enzyme, thus suggesting a double role for TOP as a peptide degrading and also as peptide-binding protein [4].TOP is ubiquitously found in the cytosol of mammalian tissues [5], suggesting that it should perform a general function in the intracellular oligopeptide metabolism. Among other possibilities, the hydrolytic and the nonhydrolytic peptide binding activity of T...