The thyroliberin and luliberin contents exhibit a diurnal rhythm within the brain and an asymmetrical distribution of both neuropeptides has been demonstrated in the hypothalamus. Since they have been described as substrates of pyroglutamyl peptidase I, this activity was analysed in several rat brain regions and other structures, in order to investigate its putative diurnal variations and changes in relation to the distribution of these neuropeptides and/or other susceptible substrates. Fluorometric activity was assayed in the retina, pituitary gland, superior cervical ganglia, pineal gland, some selected brain regions, and serum of adult male rats at six different time points of a 12:12 h light:dark cycle, using pyroglutamyl-β-naphthylamide as substrate. A significant circadian rhythm was demonstrated in the retina, the hypothalamus, and the intermediate-posterior pituitary lobe. In addition, a small but significant asymmetrical distribution of this activity, at certain time points, was disclosed: The activity was higher in the left than in the right retina at 10 h of the light period, whereas it was predominant in the right side at 01 h of the dark period. Furthermore, the activity was higher in the left anterior than in the right hypothalamic area at 13 and 01 h. These results could be indicative of a role of this enzymatic activity in the control of the functional status of its endogenous substrates.