Prolyl, cystyl and pyroglutamyl peptidases are emerging targets for diabetes and cognitive deficit therapies. The present study is focused on the influence of diabetes mellitus induced by streptozotocin on levels of representative hydrolytic activities of these enzymes in the rat hypothalamus and hippocampus. Streptozotocin-diabetic rats presented about 348mg glucose/dL blood, and a slightly increased hematocrit and plasma osmolality. The activities of soluble and membrane-bound dipeptidyl-peptidase IV, and soluble cystyl aminopeptidase did not differ between diabetic and control rats in both brain areas. Hippocampal soluble prolyl oligopeptidase presented similar activities between diabetic and controls. Increased activities in diabetics were observed for soluble prolyl oligopeptidase (1.78-fold) and membrane-bound cystyl aminopeptidase (2.55-fold) in the hypothalamus, and for membrane-bound cystyl aminopeptidase (5.14-fold) in the hippocampus. In both brain areas, the activities of membrane-bound and soluble pyroglutamyl aminopeptidase were slightly lower (<0.7-fold) in diabetics. All modifications (except hematocrit) observed in streptozotocin-treated rats were mitigated by the administration of insulin. Glucose and/or insulin were shown to alter in vitro the hypothalamic activities of soluble pyroglutamyl aminopeptidase and prolyl oligopeptidase, as well as membrane-bound cystyl aminopeptidase. These data provide the first evidence that diabetes mellitus generates direct and indirect effects on the activity levels of brain peptidases. The implied regional control of regulatory peptide activity by these peptidases suggests novel potential approaches to understand certain disruptions on mediator and modulatory functions in diabetes mellitus.