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The Upper Aniene River basin is part of a large karst aquifer, which interacts with the river, and represents the most important water resource in the southeast part of Latium Region, Central Italy, used for drinking, agriculture and hydroelectric supplies. This work provides hydrogeochemical data and their interpretations for 1 spring and 2 cross section of Aniene River, monitored from July 2014 to December 2015, in the Upper Valley of Aniene River, to identify flow paths and hydrogeochemical processes governing groundwater-surface water interactions in this region. These activities deal with the Environmental Monitoring Plan made for the catchment work project of the Pertuso Spring, in the Upper Valley of Aniene River, which is going to be exploited to supply an important drinking water network in the South part of Rome district. Discharge measurements and hydrogeochemical data were analyzed to develop a conceptual model of aquifer-river interaction, with the aim of achieving proper management and protection of this important hydrogeological system. All groundwater samples are characterized as Ca-HCO3 type. Geochemical modeling and saturation index computation of the water samples show that groundwater and surface water chemistry in the study area was evolved through the interaction with carbonate minerals. All groundwater samples were undersaturated with respect to calcite and dolomite, however some of the Aniene River samples were saturated with respect to dolomite. The analysis of Mg2+/Ca2+ ratios indicates that the dissolution of carbonate minerals is important for groundwater and surface water chemistry, depending on the hydrological processes, which control the groundwater residence time and chemical equilibria in the aquifer.