To evaluate the effects of light and darkness on pyroglutamyl peptidase I activity (pGluPI) and its left-right distribution, pGluPI was measured bilaterally in the retina and hypothalamus under selected light-dark schedules. Rats under a 12 h light-dark cycle were divided into four experimental groups. After the end of the 12 h dark period, the animals were kept two additional hours in darkness (group 1), or light (group 2). After the end of the 12 h light period, the animals were kept two additional hours in darkness (group 3), or light (group 4). Experiments were done in light or darkness depending on the 2 h period. In the retina, a previous 12 h light period led to higher values of enzyme activity than dark periods. Left-right predominance, however, depended on the previous 2 h period: the light period led to left predominance, whereas right predominance was found after the 2 h dark period. In the hypothalamus, a left predominance was found only in group 3. These results demonstrate that environmental light conditions influence pGluPI activity in the rat retina and hypothalamus.