► We examine the origin and chemical evolution of groundwater in southern Tunisia. ► Contribution of water from the deep CI aquifer to Turonian formation was demonstrated. ► Influence of geogenic carbon dioxide in the studied groundwater system was shown. ► The work aids management strategies of scarce groundwater resources in Tunisia. Groundwater is the most important source of water supply in southern Tunisia. Previous hydrogeologic and isotopic studies carried out in this region revealed the existence of two major aquifer systems: the “Complex Terminal” (CT) and the “Continental Intercalaire” (CI). Turonian carbonates constitute one of the major aquifer levels of the CT multilayered aquifer. It extends over most of southern Tunisia, and its hydrodynamic regime is largely influenced by tectonics, lithology and recharge conditions. Forty-eight groundwater samples from the CI and Turonian aquifers were collected between January and April 2004 for chemical and isotopic analyses. Hydrochemistry and isotopic tools were combined to get an insight into the processes controlling chemical composition of groundwater and wide-scale interaction of these two aquifer systems. Analysis of the dissolved constituents revealed that several processes control the observed chemical composition: (i) incongruent dissolution of carbonate minerals, (ii) dissolution of evaporitic minerals, and (iii) cation exchange. Dissolution alone cannot account for the observed high supersaturation states of groundwater with respect to calcite and dolomite. The observed supersaturation is most probably linked to geogenic CO 2 entering water-bearing horizons of the CT and CI aquifers via deep tectonic faults and discontinuities and subsequent degassing in the exploitation wells. Presence of geogenic CO 2 in the investigated region was confirmed by C isotope data of the DIC reservoir. The radiocarbon content of the Turonian samples varied between 9.5 and 43 pmc. For CI samples generally lower values were recorded, between 3.8 and 22.5 pmc. Stable isotope composition of Turonian groundwater samples varied from −8.3 to −5.3‰ for δ 18 O and from −60 to −25‰ for δ 2 H. The corresponding ranges of δ values for the Continental Intercalaire samples were from −8.9‰ to −6.9‰ for δ 18 O and from −68.2‰ to −45.7‰ for δ 2 H. Stable isotope composition of groundwater representing CT and CI aquifers provide strong evidence for regional interaction between both systems.