Elevated extracellular D-glucose concentration or gestational diabetes increase L-arginine transport via human Cationic Amino acid Transporters 1 (hCAT-1) and nitric oxide (NO) synthesis by the endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVEC)(Sobrevia et al. 1997). However, the mechanisms causing these changes in L-arginine/NO pathway are not well understood. Hyperglycaemia and diabetes mellitus are associated with increased synthesis and release of Transforming Growth Factor β1 (TGF-β1)(McGinn et al. 2003). We investigated whether the effect of D-glucose on L-arginine/NO pathway involves TGF-β1. HUVEC from normal pregnancies (Ethics Committee approval and informed patient consent were obtained) were cultured in medium 199 (M199) containing heat-inactivated foetal and newborn calf serum (20% v/v), 3.2 mM L-glutamine, 100 iu/ml penicillin-streptomycin (37°C, 5% CO₂). Prior to experiments, confluent cells were cultured for 2 h in M199 containing 2% newborn calf serum, and exposed to this medium (0-24 h) containing 5 mM D-glucose, 5 mM D-glucose + TGF-β1 (2 ng/ml), 25 mM D-glucose, or 25 mM D-glucose + TGF-β1. L-Arginine transport (100 μM, 2 μCi/ml, 37°C, 1 min) was measured and total eNOS protein levels were determined by Western blot. hCAT-1 mRNA level was quantified by real time PCR. Total and active TGF-β1 level in the supernatant was measured by ELISA. Basal L-arginine transport (2.5 ± 0.2 ρmol/μg protein/min, mean ± S.E.M., n=12) was increased (3.1-fold) by TGF-β1 (P<0.05, unpaired Student t test) at 1-6 h of incubation in 5 mM D-glucose. Incubation with 25 mM D-glucose stimulated L-arginine transport at 1-6 h (2.9-fold) and then at 18-24 h (2.8-fold). L-Arginine transport was not further increased when cells were co-incubated with 25 mM D-glucose and TGF-β1. Incubation with 25 mM D-glucose increased active (0.39 ± 0.02 vs 0.28 ± 0.02 ρg/μg protein/h) and total (0.41 ± 0.01 vs 0.26 ± 0.02 ρg/μg protein/h) TGF-β1 release from HUVEC. TGF-β1 and 25 mM D-glucose increased eNOS protein level (1.6 and 1.7-fold, respectively) and hCAT-1 mRNA level (18 and 21-fold, respectively). Thus, D-glucose-stimulated L-arginine/NO pathway could result from increased expression and activity of hCAT-1 and eNOS in response to TGF-β1 released from HUVEC.