Hyperglycemia results in various retinal metabolic abnormalities that can contribute to the development of retinopathy, but it has been difficult to recognize which abnormalities are critical. In this study, the possible interrelationship between hyperglycemia-stimulated oxidative stress, protein kinase C (PKC), and nitric oxide (NO) was investigated by examining the effects of inhibitors of oxidative stress, PKC and NO on glucose-induced retinal oxidative stress, PKC activity and NO levels concentrations, both under in vitro conditions in retinal endothelial cells and isolated retina, and in vivo in the retina from diabetic rats. Bovine retinal endothelial cells were incubated in 5 or 30 mM glucose for 3 days in the presence or absence of inhibitors of oxidative stress (N-acetyl cysteine), PKC (LY333531), or NO (L-NAME). Incubation of retinal endothelial cells in 30 mM glucose resulted in an approximately 2-fold elevation in retinal TBARS, PKC and NO. Addition of N-acetyl cysteine, LY333531, or L-NAME significantly inhibited glucose-induced elevation in oxidative stress, NO and PKC. Similar results were obtained when intact retinas from normal rats were incubated with 30 mM glucose for 6 hours. In diabetic rats, elevations in retinal TBARS, PKC and NO were observed at 2 months of diabetes, and administration of N-acetyl cysteine, LY333531 or aminoguanidine prevented diabetes-induced elevation in retinal TBARS and NO levels, and PKC activity. Thus, these results suggest that diabetes-induced metabolic abnormalities, originally considered to be independent abnormalities, are apparently interrelated in retina; inhibiting a single retinal abnormality may have multiple beneficial effects to correct retinal dysmetabolism and to inhibit the development of retinopathy.