The effect of the adenosine (AD) analog 2-chloroadenosine (C-AD) on glucose-induced inhibition of phosphoinositide synthesis was studied in human retinal pigment epithelial (RPE) cells by monitoring the level of the phosphatidylinositol (PI) synthase substrate, cytidine diphosphate diglyceride (CDP-DG). In high-aldose reductase (AR)-expressing RPE 91 cells, C-AD decreased CDP-DG at 5 mmol/L glucose and reversed the increase by 20 mmol/L glucose. AD deaminase (ADA), which inactivates endogenously released AD, potentiated the hyperglycemia-induced increase in CDP-DG. Theophylline, an AD-A1 and AD-A2 receptor antagonist, caused an increase in CDP-DG at 20 mmol/L glucose. C-AD did not alter CDP-DG in low-AR-expressing RPE 45 cells, but did decrease CDP-DG after cells were conditioned in 300 mmol/L glucose for 1 week (which induces AR). The mechanism by which AD regulates PI synthase in cells with high AR activity is unknown, but it is independent of Gi or Gs proteins, adenylate cyclase and phospholipase C (PLC) activation, myo-inositol (MI) uptake, or MI efflux. Administration of C-AD to streptozotocin-induced diabetic rats prevented the slowing of motor nerve conduction velocity (MNCV). Thus, AD derivatives, which reverse a glucose-induced deficit in phosphoinositide metabolism, might serve as a useful pharmacological tool to intervene in hyperglycemia-induced diabetic complications.