L-Arginine transport and nitric oxide (NO) synthesis are increased in human umbilical vein endothelium (HUVEC) exposed to high D-glucose (Vásquez et al. 2005). Hyperglycemia increases expression of Transforming Growth Factor β1 (TGF-β1)(McGinn et al. 2003), which stimulates endothelial NO synthase expression (Oyadomari et al. 2001). It is unclear whether TGF-β1 alters expression of L-arginine transporters, and whether TGF-β type II receptors (TβRII) are required for D-glucose stimulation on L-arginine transport. We have characterized TGF-β1 and TβRII roles on D-glucose stimulated L-arginine transport. Confluent HUVEC (M199, 20% sera, 3.2 mM L-glutamine, 100 IU/ml penicillin-streptomycin, 37°C, 5% CO₂) were exposed (2 h) to 2% sera and then 0-24 h to 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-[³H]Arginine transport (15-1000 µM, 2 µCi/ml, 37°C, 1 min) was determined. Human Cationic Amino acid Transporter 1 (hCAT-1) mRNA was quantified by real time RT-PCR (28S rRNA was internal reference). Latent and active TGF-β1 levels were measured by ELISA. Phosphorylated and total Smad2 and p42/44^mapk protein levels were determined by Western blot. A replication-defective adenoviral vector expressing a truncated human TβRII receptor (Ad-TTβRII) was prepared (Yamamoto et al. 1996) and HUVEC were transfected (2% serum, 12 h). Maximal velocity (V_max) of L-arginine transport (V_max 6.9±0.1 pmol/μg protein/min, K_m 113±5 μM, mean±SEM, n=23) is increased (P<0.05, unpaired Student’s t test) by 25 mM D-glucose (V_max 17±1 pmol/μg protein/min, K_m 140±24 μM) or TGF-β1 (V_max 19±1 pmol/μg protein/min, K_m 123±25 μM). D-Glucose stimulation of L-arginine transport was unaltered (P>0.05) by TGF-β1 (V_max 15±0.8 pmol/μg protein/min, K_m 147±23 μM). L-Arginine (100 μM) transport was unaltered (P>0.05) in cells overexpressing TTβRII (Control 2.8±0.3, 25 mM D-glucose 4.2±0.6, TGF-β1 4.5±0.4 pmol/μg protein/min). hCAT-1 mRNA expression (3.1 x10⁴ mRNA copies) was increased by high D-glucose (6.7-fold) and TGF-β1 (4.6-fold) in not transfected cells. Smad2 and p42/44^mapk phosphorylation were increased by high D-glucose (3.8-fold) and TGF-β1 (4.1-fold); however, total protein was unaltered. Smad2 and p42/44^mapk phosphorylation was blocked by PD-98059 and was negligible in cells overexpressing TTβRII. Latent (0.51±0.06 ng/ml/10⁶ cells) and active (0.49±0.07 ng/ml/10⁶ cells) TGF-β1 in 25 mM D-glucose were higher than control (latent 0.28±0.04, active 0.21±0.03 ng/ml/10⁶ cells). Thus, high D-glucose–stimulated L-arginine transport could result from a mechanism involving TβRII stimulation by TGF-β1, involving activation of p42/44^mapk and Smad2.