The design and development of experimental, in vivo, chronobiological animal models may help reveal some of the relationships between circadian rhythms and biological functions. In vivo experiments require the use of appropriate anesthesia, which should be selected according to their particular effect on the organism. The aim of study was to review the status of acid-base balance and ion concentration in arterial blood under common used general anesthesias in experiments in dependence on the light-dark (LD) cycle in spontaneously breathing rats. The experiments were performed using 3-to 4-monthold pentobarbital(P)-, ketamine/xylazine(K/X)-, and zoletil(Z)-anesthetized female Wistar rats after a 4-week adaptation to an LD cycle (12 h light and 12 h dark). We concluded that P anesthesia disturbs LD dependence of acid-base balance compared to K/X and Z anesthesia, but LD differences in plasma ion concentrations are disturbed under all type of general anesthesia. P anesthesia is not the most appropriate type of anesthesia in rat chronobiological experiments. It eliminated LD differences and also produces a more acidic environment, more pronounced hypercapnia and hypoxia than K/X and Z anesthesias. This should be taken into account because the altered internal environment may affect the activity of systems whose functions are primarily dependent on acid-base balance.