The endogenous nucleoside adenosine causes vasodilatation of human placenta vasculature [1] via a mechanism involving increased L-arginine uptake via cationic amino acid transporters 1 (hCAT-1) due to A2A adenosine receptors (A2AAR) activation in human umbilical vein endothelial cells (HUVEC) [2]. hCAT-1 activity and expression is increased by insulin in HUVEC [3], and A2AAR activation increases insulin sensitivity in subjects with insulin resistance [4]; however, a potential A2AAR involvement in L-arginine transport modulation by insulin in HUVEC is unknown [5]. The aim of this study was todetermine whether insulin-stimulation of hCAT-1-mediated L-arginine transport involves A2AAR in HUVEC. Methods: Primary cultured HUVEC (passage 2) from full-term normal pregnancies were used. Insulin (1 nM, 8 hours) was assayed on hCAT1 expression (SLC7A1 promoter activity (firefly/renilla luciferase reporter activity for pGL3-hCAT1-1606 and pGL3-hCAT1-650 constructs), mRNA expression (quantitative real time PCR), protein abundance (Western blot)) and L-arginine transport (0-1000 µM L-arginine, 3 µCi/ml L-[3H]arginine, 1 minute, 37 °C). Assays were done in absence or presence of 4-(2-[7-amino-2-[2-furyl]-[1,2,4]triazolo[2,3-a]{1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM-241385, 10 nM, A2AAR antagonist), 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5`-N-ethylcarboxamidoadenosine (CGS-21680, 30 nM, A2AAR agonist) and/or S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 10 µM, adenosine transport inhibitor). Results: Insulin and NBTI increased (P<0.05, ANOVA, n=5-10 different cell cultures) extracellular adenosine concentration, the maximal velocity (but not the apparent Km) for L-arginine transport, and hCAT-1 expression (protein and mRNA). These effects were blocked by 10 nM ZM-241385. ZM-241385-inhibited SLC7A1 reporter transcriptional activity was similar in cells transfected with pGL3-hCAT-1-1606 or pGL3-hCAT-1-650 constructs, and comparable to the activity determined for pGL3-hCAT-1-650 construct in presence of NBTI + insulin. However, reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1-1606, and the ZM-241385 sensitive fraction of NBTI response was similar in absence or presence of insulin. Conclusion: hCAT-1 expression and activity is under regulation by insulin via a mechanism requiring functional A2AAR in HUVEC. These findings could be determinant in diseases associated with fetal insulin resistance, such as gestational diabetes.