Vitamin K-1 (VK1) was shown by voltammetry and coulometry to undergo two chemically reversible one-electron reduction processes in acetonitrile (CH3CN) containing 0.2 M Bu4NPF6 as the supporting electrolyte. The potential separation between the first and second electron-transfer steps diminished sequentially with the addition of water, so that at a H2O concentration of approximately 7 M (similar to 13% v/v) only one process was detected, corresponding to the reversible transfer of two electrons per molecule. The voltammetric behavior was interpreted on the basis of the degree of hydrogen bonding between the reduced forms of VK1 with water in the solvent. It was found that the potential separation between the first and second processes was especially sensitive to water in the low molar levels (0.001-0.1 M); therefore, by measuring the peak separation as a function of controlled water concentrations (accurately determined by Karl Fischer coulometric titrations) it was possible to prepare calibration curves of peak separation versus water concentration. The calibration procedure is independent of the type of reference electrode and can be used to determine the water content of CH3CN between 0.01 and 5 M, by performing a single voltammetric scan in the presence of 1.0 mM VK1. The voltammetry was also investigated in dichloromethane, dimethylformamide, and dimethyl sulfoxide. The reduction processes were monitored by in situ electrochemical UV-vis spectroscopy in CH3CN over a range of water concentrations (0.05-10 M) to spectroscopically identify the hydrogen-bonded species.