Purines are responsible for the regulation of many physiological and pathological processes in the central nervous system including the development and remodeling of tissues after trauma, stress, ischemia, and neurodegenerative diseases. The extracellular concentration of purines significantly increases under conditions of stress. We investigated the effects of purine nucleosides and nucleobases on primary cultures of Wistar rat astrocytes not only under physiological conditions, but also after deprivation of energy depots by using iodoacetic acid, and assessed whether the investigated molecules exhibited cytoprotective or cytotoxic effects. An MTT test was used to evaluate cell viability, whereas flow cytometry analysis was employed to evaluate the presence of free oxygen species. Survival of astrocytes (as evidenced by the MTT test) decreased on treatment with 500 µM iodoacetic acid for 40 min (to 58.4% compared with untreated controls). The cytotoxic effect of iodoacetic acid was partially reversed by co-treatment with either 200 µM adenosine (to 80.3% of control) or 200 µM guanosine (to 83.3% of control), whereas the application of inosine or adenine had no effect. Iodoacetic acid alone did not increase the production of free oxygen radicals, but the induction of oxidative stress was observed during the concurrent incubation of astrocytes with 200 µM adenosine and 500 µM iodoacetic acid. We conclude that, under energy deprivation, adenosine and guanosine stimulate the survival of astrocytes by means of the recovery of intracellular ATP.