The aim of this study was to characterize the mechanism of the chemical interaction between L-ascorbic acid (ASC) and tetrahydrobiopterin (BH(4)) in vitro and to examine its effect on the activity of endothelial nitric oxide synthase (eNOS) in first trimester human placentae. At room temperature, in Tris-HCl buffer (pH 7.4), both ASC and BH(4) were readily oxidized by dissolved O(2) or H(2)O(2). BH(4) was more sensitive to auto-oxidation, while ASC was more susceptible to oxidation by H(2)O(2). Addition of 36 micromol/l BH(4) to 143 micromol/l ASC increased the initial rate of ASC oxidation 3.2-fold in a catalase-sensitive manner, indicating that enhanced ASC oxidation is partly due to the formation of H(2)O(2). In the presence of catalase, BH(4) still stimulated 1.9-fold the initial rate of ASC oxidation, suggesting that another auto-oxidation product of BH(4), most probably quininoid-BH(2) (qBH(2)), could also stimulate ASC oxidation while itself being reduced back to BH(4). ASC prevented the auto-oxidation of BH(4) in a concentration-dependent fashion, with 3 mmol/l ASC providing an almost complete stabilization of 25 micromol/l BH(4). Importantly, basal eNOS activity in placental microsomes was stimulated 2.5-fold by 0.5 micromol/l BH(4), and 0.5 mmol/l ASC enhanced the BH(4)-stimulation 1.4-fold, with a smaller effect on basal eNOS activity. Taken together, the findings support the notion that the stabilizing action of ASC on BH(4) is related to the ASC-mediated reductive reversal of the auto-oxidation process of BH(4). Moreover, we demonstrated that concentrations of ASC present in the placenta as a common vitamin C supply are sufficient to protect cellular free BH(4) and may contribute to the stimulation of placental eNOS activity.