Adenosine, produced from the decomposition of adenosine triphosphate, is believed to provide protective effects during ischemia. On the other hand, adenosine metabolites may serve as precursors for oxygen free radical formation. These substances have not been previously measured in intact vertebrate retina, where adenosine and its metabolites may play a role in the pathogenesis of ischemic injury. The small tissue mass of the retina, particularly in rats, renders these measurements challenging. Furthermore, accurate measurement of purine nucleosides requires immediate cessation of ongoing adenosine metabolism. Concentrations of adenosine and its purine nucleoside metabolites inosine, hypoxanthine, and xanthine in the retina of ketamine/xylazine-anesthetized rats were measured after in situ freezing using high-performance liquid chromatography. The retina was removed from the frozen eyes and analyzed. Quantitative measurements were made possible through the use of an internal standard. Ischemia was induced by ligation of the central retinal artery. Retinal purine nucleoside concentrations did not differ between the two eyes of the rat under control conditions, and there was no effect of placement of the ligating suture itself compared to completely unmanipulated eyes. Use of two different in situ freezing methods yielded comparable results. To evaluate the impact of a period of ischemia, one retina of each rat was ischemic for 30 min, and the other, non-ischemic. Our measurements were associated with a high degree of reproducibility and minimal variability, and significant changes in purine nucleoside concentrations were detectable in the retina after 30 min of ischemia. Our method may be used to assess the role of adenosine and its metabolites in the pathogenesis of ischemic neuronal injury, including in the retina.