Hyperglycemia in diabetes mellitus has been shown to activate diacylglycerol (DAG)-protein kinase C (PKC) pathway in the vascular tissues, possibly altering vascular function. We have characterized the effects of vitamin E (d-alpha-tocopherol) on activation of PKC and DAG levels in retinal tissues of diabetic rats, and correlated its effects to retinal hemodynamics using video-based fluorescein angiography (VFA). Comparing streptozotocin-induced diabetic rats to controls, membranous PKC specific activities were increased by 71% (p < 0.05). Western blot analysis showed that the membranous PKC beta II isoform was significantly increased by 133 +/- 45% (p < 0.05). Intraperitoneal injection of d-alpha-tocopherol (40 mg/kg) every other day prevented the increases in membranous PKC specific activity and PKC beta II protein shown by immunoblots. Similar to PKC activities, total DAG levels were increased in the retina and were normalized by d-alpha-tocopherol treatment. Physiologically, abnormalities of retinal blood hemodynamics, as measured using VFA, which previously have been reported to be associated with increases of DAG and PKC levels in the diabetic rats, were prevented by d-alpha-tocopherol treatment in diabetic rats. The direct effect of d-alpha-tocopherol on total DAG and [3H]-palmitate incorporation into DAG were also examined using cultured bovine retinal endothelial cells (REC). Exposure of REC to 22 mM glucose for three days increased total DAG and [3H]-palmitate labeled DAG levels by 35 +/- 8% and 50 +/- 8%, respectively (p < 0.05). The presence of d-alpha-tocopherol (50 micrograms/ml) prevented the increase of both total DAG and [3H]-palmitate labeled DAG levels in cells exposed to 22 mM glucose. These findings suggested that the mechanism of the d-alpha-tocopherol's effect appears to be mediated by the normalization of the hyperglycemia-induced activation of the DAG-PKC pathway which leads to the normalization of abnormal retinal blood flow seen in diabetes mellitus.