Barbiturates are regularly used as an anesthetic for animal experimentation and clinical procedures and are frequently provided with solubilizing compounds, such as ethanol and propylene glycol, which have been reported to affect brain function and, in the case of 1H NMR experiments, originate undesired resonances in spectra affecting the quantification. As an alternative, thiopental can be administrated without any solubilizing agents. The aim of the study was to investigate the effect of deep thiopental anesthesia on the neurochemical profile consisting of 19 metabolites and on glucose transport kinetics in vivo in rat cortex compared with α-chloralose using localized 1H NMR spectroscopy. Thiopental was devoid of effects on the neurochemical profile, except for the elevated glucose at a given plasma glucose level resulting from thiopental-induced depression of glucose consumption at isoelectrical condition. Over the entire range of plasma glucose levels, steady-state glucose concentrations were increased on average by 48% ± 8%, implying that an effect of deep thiopental anesthesia on the transport rate relative to cerebral glucose consumption ratio was increased by 47% ± 8% compared with light α-chloralose-anesthetized rats. We conclude that the thiopental-induced isoelectrical condition in rat cortex significantly affected glucose contents by depressing brain metabolism, which remained substantial at isoelectricity.