De novo biosynthesis of myo-inositol (MI) by permeabilized cultured bovine retinal capillary pericytes (BRCP) and feline retinal pigment epithelial cells (FRPE), grown in different concentrations of glucose, were studied. After incubation with a physiological concentration of [14C]glucose 6-phosphate (G6P), the radioactive G6P derivatives were quantitated by a single HPLC column. Based on the determined specific activity of [14C]G6P, activities of inositol 1-phosphate synthase (MI synthase) were calculated. The activity of MI synthase was reduced 48% by growing BRCP in a high-glucose medium (20 mM) in comparison with that in the normal medium (glucose 5 mM). In contrast, the de novo MI biosynthesis by FRPE was not changed with increasing concentrations of glucose in the medium. As compared with MI uptake previously studied, the synthesized MI contributes a substantial proportion of cellular MI pool in BRCP. Therefore, in BRCP growing in high glucose the reduced MI biosynthesis aggravates the low MI content resulting from the inhibited MI uptake, and thus leads to altered inositol phospholipid metabolism.