The formation of inositol phospholipids (IPLs) and inositol phosphate esters (IPEs) in response to glucose was studied in isolated retinal microvessels from porcine eyes. Retinal microvessels incubated from 60 hr with myo-[3H]inositol were sequentially extracted to obtain IPLs and IPEs. [3H]Inositol-labelled IPLs were deacylated to produce the corresponding glycero derivatives. Both deacylation products and water-soluble IPEs were monitored by anion-exchange chromatography. In the presence of high glucose (30 mM) the labelling in inositol triphosphate (IP3) was reduced to 77% and was restimulated by adding myo-inositol (final concentration 0.4 mM) to 158% of the control under physiological conditions of glucose (5 mM) and myo-inositol (0.04 mM). With a fixed glucose concentration (5 mM), IPE accumulation was observed with increasing concentrations of exogenous myo-inositol. Under physiological conditions (glucose 5 mM, myo-inositol 0.04 mM) the distribution (percentage) of radioactivity in phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) was 63:19:18. The myo-inositol concentration dependence of IPL formation was also demonstrated. A decrease in IP3 in response to high glucose without changing PIP2 but with a reduction in PI indicated that PI may act as a reservoir to replace a possible loss of PIP2. These findings suggest that availability of myo-inositol by retinal microvessels may be essential to maintain the normal signal transduction and cell proliferation associated with IPL turnover under high glucose concentration.