Nitric oxide (NO) is synthesized from the cationic amino acid L-arginine. Transport of L-arginine occurs mainly via the family of cationic amino acid transporters (CAT), system y⁺/CAT, and via system y⁺L (Deves & Boyd, 2000). System y⁺L exhibit very high affinity, exchanges cationic by neutral amino acids in presence of Na⁺, and is a heterodimer formed by a heavy chain (4F2hc) and a light chain (4F2-lc2 or 4F2-lc3) of the surface antigen 4F2. Elevated D-glucose modulates system y⁺/CAT activity and expression in human umbilical vein endothelium (HUVEC), however there are not reports regarding modulation of system y⁺L by D-glucose. We determined whether expression and activity of system y⁺L is modulated by D-glucose and its potential role in the regulation of L-arginine/NO pathway in HUVEC. Cells were cultured in medium 199 containing 20% bovine sera, 3.2 mM L-glutamine, 100 iu/ml penicillin-streptomycin (37°C, 5% CO₂). Passage 2 cells were exposed (0-24 h) to medium containing 5-25 mM D-glucose. L-Arginine transport (L-[³H]arginine, 0.1-20 μM, 4 μCi/ml, 37°C, 1 min) was measured in absence or presence of L-leucine (1 mM), N-ethylmaleimide (NEM, 200 μM, system y⁺/CATs inhibitor), 12-myrystate 13-acetate [PMA, 100 nM, protein kinase C (PKC) activator], calphostin C (100 nM, PKC inhibitor) or PD-98059 [10 μM, MAP kinase kinase 1 and 2 (MEK1/2) inhibitor]. PKC activity was monitored by phosphorilation of a PKC substrate. Experiments were done in Krebs solution with or without Na⁺. SLC3A2/4F2hc, SLC7A7/4F2-lc2 and SLC7A6/4F2-lc3 genes encoding for system y⁺L were detected by RT-PCR. Transport of 1.5 μM L-arginine was inhibited (P<0.05, unpaired Students t test, n=8-12) by L-lysine, L-leucine and L-phenylalanine, but was unaltered by L-alanine or L-cysteine. NEM did not alter 1.5 μM L-arginine transport, but inhibited (92 ± 3%, mean ± S.E.M.) transport of 100 μM L-arginine. L-Arginine transport in presence of NEM was saturable (V,_max 0.4 ± 0.03 pmol/μg protein/min; K_m, 1.7 ± 0.2 μM) and competitively inhibited by L-leucine in presence of Na⁺ (V_max, 0.51 ± 0.07 pmol/μg protein/min; K_m, 6.6 ± 1.1 μM). N^G-Nitro-L-arginine methyl ester and L-leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 μM L-arginine. Cells exposed to high D-glucose or PMA exhibited increased PKC activity, but reduced transport activity of system y⁺ L and NO synthesis, effects blocked by calphostin C. PD-98059 did not alter the effect of D-glucose, but inhibited L-arginine transport in normal D-glucose. Elevated D-glucose increased p42/44^mapk phosphorylation. In conclusion, L-arginine transport via system y⁺ L is inhibited by D-glucose through activation of PKC, an effect that seems not to involve MEK1/2. However, MEK1/2 could act as a tonic activator of system y⁺ L in HUVEC in normal D-glucose.