Pyroglutamyl aminopeptidase (pGluAP) is an omega peptidase that hydrolyzes biologically active peptides, such as thyrotropin-releasing hormone (TRH), with neuronal and extraendocrine functions. We analyzed the effects of a cholesterol-enriched diet on soluble and membrane-bound pGluAP activity in frontal cortex, pituitary and adrenal glands of male and female mice using fluorimetric assays. Significant increases were observed in soluble pGluAP activity in the frontal cortex and adrenal glands in males and in the pituitary in females. Membrane-bound pGluAP activity was increased in the frontal cortex and pituitary of males and females after the mice were fed a cholesterol-enriched diet. These increases may produce changes in the metabolism of endogenous substrates, including TRH, which may be related to alterations in its neuromodulator functions and to the possible relationship between TRH and other neurotransmitter systems.