We studied changes in water chemistry along the groundwater flow path in the Matsumoto tunnel vicinity and estimated the origin of the groundwater and dissolued substances. The relationship between the concentration of HCO₃^-, Ca²⁺, and Na⁺, and the CO₂ gas pressure in the groundwater indicated that the HCO₃^-, Ca²⁺, and Na⁺ were produced by reaction of the CO₂ gas in the groundwater and feldspar in the rocks. The relationship between the concentration of NO₃^- and the Eh and pH values in the groundwater indicated that in an oxidative condition, some bacteria used NH₄⁺ and produced NO₃^- and H⁺ and in a reductive condition, other bacteria used NO₃^- and produced N₂ gas and OH^-. The stable hydrogen and oxygen isotopic ratios in the groundwater and precipitation indicated that the groundwater originated from precipitation fell on the area. The concentration of ³H and the stable hydrogen and oxygen isotopic ratios in the groundwater suggested that it was getting warmer for more than 60 years. The stable carbon isotopic ratio indicated that the HCO₃^- in the groundwater originated from CO₂ gas produced by organic matter in the soil except in the deep well water. The deep well water, which had a higher concentration of HCO₃^- than the other groundwater sampled was thought to have acquired HCO₃^- through contact with rocks. The stable sulfur isotopic ratio indicated that the SO₄^2- in the groundwater was of the same origin.