The myo-inositol transport system in confluent fetal-bovine aortic endothelial cells was characterized after 7-10 days in subculture, at which time the myo-inositol levels and rates of myo-[2-3H]-inositol uptake and incorporation into phospholipid had reached steady state. Kinetic analysis indicated that the uptake occurred by both a high-affinity transport system with an apparent Kt of 31 microM and Vmax. of 45 pmol/min per mg of protein, and a non-saturable low-affinity system. Uptake was competitively inhibited by phlorhizin, with a Ki of 50 microM; phloretin was a non-competitive inhibitor, with half-maximal inhibition between 0.2 and 0.5 mM. Glucose was a weak competitive inhibitor, with a Ki of 37 mM; galactose failed to inhibit uptake. A weak dependence on Na+ for the initial rate of uptake was observed at 11 microM myo-inositol. When fetal-bovine-serum (FBS)-supplemented medium, which contained 225 microM myo-inositol, was used, the cells contained about 200 nmol of myo-inositol/mg of DNA. With adult-bovine-serum (ABS)-supplemented medium, which contained 13 microM myo-inositol, the cells contained about 110 nmol/mg of DNA. Transport of 11 microM myo-[2-3]inositol was 18 and 125 pmol/min per mg of DNA for cells grown in FBS and ABS respectively. Kinetic analysis showed that for the cells grown in FBS the Vmax. of the high-affinity system was decreased by 64%, whereas the Kt remained essentially unchanged. Increased cell myo-inositol levels were not associated with an increased rate of phosphatidylinositol synthesis. After prolonged exposure of fetal endothelial cells to a myo-inositol concentration which approximated to a high fetal as opposed to a low adult blood level, cell myo-inositol levels doubled and high-affinity transport underwent down-regulation.