Introduction: Human umbilical vein endothelial cells (HUVECs) from gestational diabetes mellitus (GDM) pregnancies show reduced adenosine transport via the human equilibrative nucleoside transporters 1 and 2 (hENT1/2) (Westermeier et al., 2011). Increased nitric oxide (NO) generation is seen in an alkaline intracellular medium, and high NO reduced the hENT1/2 expression and activity in HUVECs. Furthermore, GDM associates with intracellular alkalization due to a higher activity of the Na+/H+ exchanger 1 (NHE1) (Fuentes et al., 2019). Aim: To evaluate whether GDM alters hENT1/2 transport activity due to intracellular alkalization. Materials & Methods: HUVECs were isolated (collagenase digestion) from full-term normal (n = 11) or GDM (n = 8) pregnancies collected at the Clinical Hospital CHRISTUS-UC (Chile) and conformed to the principles outlined in the Declaration of Helsinki. HUVECs were cultured (passage 3) in medium 199 plus sera (20%) under standard conditions (5% O2, 5% CO2). The pHi was measured in cells loaded with the fluorescent probe 2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM, 12 µmol/L, 10 min) and exposed to ammonium chloride (NH4Cl, 20 mmol/L). Basal and pHi recovery rate (dpHi/dt) were estimated (up to 360 s) in cells exposed to 5 µmol/L 5-N,N-hexamethylene-amiloride (HMA, Na+/H+ exchangers (NHE) general inhibitor), 0.1 µmol/L zoniporide (Zn, NHE1 inhibitor), 0.1 µmol/L concanamycin A (V-ATPases inhibitor), or 10 µmol/L Schering (H+/K+-ATPase inhibitor). Uptake of 2,3-[3H]adenosine (0-500 µmol/L, 3 µCi/mL, 37°C, 10 s after NH4Cl removal) was measured in the absence or presence of 1 or 10 µmol/L S-(4-nitrobenzyl)-6-thio-inosine (NBTI), inhibitory concentrations for hENT1 or hENT1+hENT2 transport, respectively. The difference between adenosine uptake in the presence of 1 and 10 µmol/L NBTI corresponded to the hENT2-mediated uptake. Results: HUVECs from GDM showed higher basal pHi compared with normal pregnancies (pHi = 7.7 ± 0.2 vs 7.1 ± 0.1, respectively) (mean ± SEM, unpaired ANOVA, P<0.05 as significant). The dpHi/dt in GDM was higher (4.2 ± 0.2 fold) than in normal pregnancies. Zn and HMA reversed the GDM-increased dpHi/dt to values in normal pregnancies. The reduced hENT1-mediated adenosine uptake in GDM at basal pHi was reversed by Zn but increased (2.2 ± 0.7 fold) in the presence of NH4Cl + Zn. However, hENT2-mediated uptake was increased (2.9 ± 0.3) in an acidic intracellular medium compared with basal pHi, but unaltered by Zn. hENT1-mediated transport was increased in the presence of NH4Cl in cells from normal pregnancies. Conclusion: NHE1-mediated alkaline pHi inhibits hENT1 and hENT2-adenosine transport in HUVECs from GDM pregnancies.