Pyroglutamyl aminopeptidase (pGluAP) is an omega peptidase which removes pyroglutamyl N-terminals residues from peptides and arylamidase derivatives. This activity is thought to be involved in the regulation of several physiological mechanisms on the central nervous system. pGluAP can modulate various susceptible endogenous substrates such as thyrotrophin-releasing hormone (TRH). It is well known that TRH plays an important role in the modulation of the behavioral changes induced by ethanol and others drugs. The aim of this work was to study the in vitro effects of ethanol (25, 50 and 100 mM) on the pGluAP activity and its ability for modulating the TRH.pGluAP activity was measured in synaptosomes from cerebral cortex of mouse, using pyroglutamyl-beta-naphthylamide as substrate in basal and stimulated (K+ 25 mM) conditions, and in presence or absence of calcium on the buffer.In basal conditions, ethanol produced an inhibition of the pGluAP activity in presence or absence of calcium, being this inhibition non dose-related. However, the stimulation with K+ 25 mM did not produce a modification of pGluAP activity in presence of calcium, but produced a light increase in absence of it. Depolarization in presence or absence of calcium and ethanol produced an inhibition of pGluAP activity, which changed in function of the ethanol concentration used.Ethanol modifies pGluAP activity in basal conditions by a mechanism independent of calcium, but the changes observed after the stimulation with high K+ may be due to a calcium-dependent mechanism. These variations of pGluAP activity induced by ethanol, and their effects on their endogenous substrates, specially TRH, may be responsible for the behavioral changes associated to the alcoholism and mediated by TRH.