Gestational diabetes is associated with increased L-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial L-arginine/NO pathway via A₂ purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter 1 (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 µM), and intracellular L-arginine (1.9 mM) and L-citrulline (0.7 mM) levels compared with normal cells (0.05 µM, 0.89 mM, 0.35 mM, respectively). Incubation of HUVEC from normal pregnancies with 1 µM nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and p42/44^mapk activation, and were blocked by the A_2a purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases L-arginine/NO pathway involving activation of MAP kinases, PKC and NO cell signalling cascades following activation of A_2a purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation of L-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus.