Ascorbic acid (AscH(2)) and dihydronicotinamide adenine dinucleotide (NADH) are two very important natural redox cofactors, which can be used as hydride, electron, and hydrogen atom sources to take part in many, important bio-reduction processes in vivo. The differences of the two natural reducing agents as hydride, hydrogen atom, and electron donors in thermodynamics, kinetics, and mechanisms were examined by using 5,6-isopropylidene ascorbate (iAscH(-)) and beta-D-glucopyranosyl-1,4-dihydronicotinamide acetate (GluNAH) as their models, respectively. The results show that the hydride-donating ability of iAscH- is smaller than that of GluNAH by 6.0 kcal/mol, but the electron-donating ability and hydrogen-donating ability of iAscH(-) are larger than those of GluNAH by 20.8 and 8.4 kcal/mol, respectively; which indicates that iAscH(-) is a good electron donor and a good hydrogen atom donor, but GluNAH is a good hydride donor. The kinetic intrinsic barrier energy of iAscH(-) to release hydride anion in acetonitrile is larger than that of GluNAH to release hydride anion in acetonitrile by 6.9 kcal/mol. The mechanisms of hydride transfer from iAscH(-) and GluNAH to phenylxanthium perchlorate (PhXn(+)), a well-known hydride acceptor, were examined, and the results show that hydride transfer from GluNAH adopted a one-step mechanism, but the hydride transfer from iAscH(-) adopted a two-step mechanism (e-H(center dot)). The thermodynamic relation charts (TRC) of the iAscH(-) family (including iAscH(-), iAsCH(center dot), iAsc(center dot-), and iAsc) and of the GluNAH family (including GluNAH, GluNAH(center dot+), GluNA(center dot), and GluNA(+)) in acetonitrile were constructed as Molecule ID Cards of iAscH(-) and of GluNAH in acetonitrile. By using the Molecule ID Cards of iAscH(-) and GluNAH, the character chemical properties not only of iAscH(-) and GluNAH but also of the various reaction intermediates of iAscH(-) and GluNAH all have been quantitatively diagnosed and compared. It is clear that these comparisons of the thermodynamics, kinetics; and mechanisms between iAscH(-) and GluNAH as hydride and electron donors in acetonitrile should be quite important and valuable to diagnose and understand the different roles and functions of ascorbic acid and NADH as hydride, hydrogen atom, and electron sources in vivo.