The hypothalamus is involved in the regulation of rhythms, autonomic, endocrine, and behavioral functions and may also participate in aging development and control. The aim of this work was to study the expression of calbindin (CB) and calretinin (CR) in the ventromedial (VMH) and dorsomedial (DMH) hypothalamic nuclei in young and old rats of both sexes by immunohistochemistry and western blotting. In young animals, the largest number of CB-immunoreactive (IR) neurons was detected in the ventral part of DMH (DMHv) and smaller percentage was found in its dorsal part (DMHd), in the dorsomedial part of the VMH (VMHdm) and in the ventrolateral part of the VMH (VMHvl). In aged animals, the percentage of CB-IR neurons significantly decreased in all studied nuclei, including DMHv, DMHd, VMHdm and VMHvl. CR-IR neurons were found in moderate number in the DMHv, DMHd, VMHdm and VMHvl of young rats. In aged rats, the percentage of CR-IR neurons significantly increased in the DMHv, DMHd, VMHdm and VMHvl. Less than one third of IR neurons colocalized CB and CR in young and aged rats. The expression of CB significantly decreased, and the expression of CR significantly increased in the DMH and VMH during aging by western blot analysis. Thus, there are opposite changes of the calcium-binding proteins expression in the hypothalamic nuclei involved in the metabolic and sexual regulation during aging.