An in situ water sampling experiment was performed in the Opalinus Clay formation (Switzerland), with the aim of obtaining undisturbed pore water samples for its characterization. The study was made from a dedicated borehole, named BDI-B1, drilled in March 2002 in the DI niche of the Mont Terri Rock Laboratory, located at the north-western part of the formation, a few meters away of the underlying Jurensis Marl formation. Five water sampling campaigns have been completed, and on site measurements of the key parameters of the water, such as pH, Eh, Fe(II), S 2− and alkalinity, were performed under controlled conditions inside an anoxic glove box. The chemical composition of the seepage waters obtained from the borehole is Na–Cl type, with an ionic strength of about 0.4 M. The Cl concentrations fit the concentration profile of the Opalinus Clay pore water obtained in previous experiments from boreholes and squeezed water samples. The highest salinity is found in this zone of the Opalinus Clay, with around 12 g/L of chloride. A perturbation of the rock system was produced during the first stages of the experiment due to a packer failure. As a consequence, the borehole was exposed to air during the first phase of the experiment. The main perturbations induced were: (1) pyrite oxidation that caused an increase of sulphate, calcium, magnesium and bicarbonate content in the waters; and (2) the inflow of 3H-bearing water vapour that could penetrate the EDZ. This fresh water infiltration could have mixed with the original formation water, and tritium contents of up to 3.8 TU were measured in the first water sampling campaigns. Nevertheless, after some time the hydrogeochemical conditions of the formation were recovered, and the long-term instrumentation and monitoring of the borehole made possible to obtain different parameters of the formation. Successive water sampling campaigns show a tendency to the stabilization of the main parameters of the water, such as sulphate and pH to values of 1600 mg/L and 7.6, respectively. The on site determination of the key parameters of the pore waters such as pH, Fe(II) and alkalinity has allowed to model the main water–rock interaction processes of the Opalinus Clay formation. A redox potential of −196 mV at pH 7.6 was obtained.