Adenosine is a vasodilator in most vascular beds, an effect depending on its extracellular concentration which is efficiently regulated by nucleoside membrane transporters. Uptake of this nucleoside in human umbilical vein endothelial cells (HUVEC) is achieved by the human equilibrative, Na⁺-independent nucleoside transporters 1 (hENT1) and hENT2 (San Martín & Sobrevia, 2006). hENT1 expression and transport activity are reduced in HUVEC isolated from pregnancies with gestational diabetes, an effect associated with increased L-arginine uptake and nitric oxide (NO) synthesis (Vásquez et al. 2004). However, regulatory mechanisms at transcriptional or post-transcriptional level accounting for this effect of gestational diabetes have not been reported. We studied the involvement of NO in the regulation of hENT1 expression and activity in HUVEC. Adenosine transport ([³H]adenosine, 2 μCi/ml, 5 s, 22°C) was measured in passage 2 cells in medium 199 (3.2 mM L-glutamine) in the absence or presence of nitrobenzylthioinosine (NBMPR, 0.1-10 μM), hypoxanthine (2 mM), N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, 10 μM, NO donor) or in cells infected with an adenovirus containing a siRNA sequence targeting eNOS (Ad-eNOS). hENT1 mRNA was determined by RT-PCR, and protein abundance by Western blot. Four fragments of upstream region of SLC29A1 gene (for hENT1) up to -3198, -2154, -1114 and -795 bp from ATG were cloned into pGL3-Basic and transfected by electroporation (320 V, 30 ms). The maximal velocity (V_max) of hENT1-mediated adenosine transport was reduced in HUVEC from gestational diabetes compared with normal cells (0.69 ± 0.1 vs 1.37 ± 0.1pmol/μg protein/s, respectively; P=0.0003, unpaired Student’s t test, mean ± S.E.M., n=7). This effect of gestational diabetes was blocked (P=0.001) by L-NAME (V_max = 2.19 ± 0.22 pmol/µg protein/s). SNAP reduced adenosine transport in normal cells (0.49 ± 0.06 pmol/µg protein/s), but not in diabetic cells (0.50 ± 0.05 pmol/µg protein/s). A reduction (60 ± 5%) of hENT1 protein abundance was found in diabetic cells, an effect blocked by L-NAME and mimicked by SNAP (67 ± 10%) in normal cells. Infection with Ad-eNOS increased hENT1 mRNA level (1.8- and 3-fold), protein abundance (2- and 4-fold) and adenosine uptake (1.8- and 2.8-fold) in normal and diabetic cells, respectively, compared with respective controls. Transcriptional activity of SLC29A1 promoter was increased (1.4-fold) by transfection of pGL3-hENT1^-1114, but was reduced (48 ± 4%) by pGL3-hENT1^-2154 in HUVEC from gestational diabetes. Reduced promoter activity by pGL3-hENT1^-2154 was blocked by L-NAME. Thus, reduced adenosine transport in gestational diabetes may result from a lower transcriptional SLC29A1 promoter activity through a NO-dependent mechanism.