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High arsenic concentrations in groundwater have been documented in La Laguna Region (LLR) in arid northern Mexico, where arsenic poisoning is both chronic and endemic. A heated debate has continued for decades on its origin. LLR consisted of a series of ancient connected lakes that developed at the end of a topographic depression under closed basin conditions. This study addresses the isotopic, chemical composition of the groundwater and geochemical modeling in the southeasternmost part of the LLR to determine the origin of arsenic. Groundwater samples were obtained from a carbonate and granular aquifers and from a clayey aquitard at terminal Viesca Lake. Results show that groundwater originated as meteoric water that reached the lakes mainly via abundant springs in the carbonate aquifer and perennial flooding of the Nazas-Aguanaval Rivers. Paleo-lake water underwent progressive evaporation as demonstrated by the enrichment of delta O-18, delta H-2 and characteristic geochemical patterns in the granular aquifer and aquitard that resulted in highly saline (> 90,000 mS/cm), arsenic-rich (up to 5000 mu g/L) paleo-groundwater (> 30,000 years BP). However, adsorption or co-precipitation on iron oxides, clay-mineral surfaces and organic carbon limited arsenic concentration in the groundwater. Arsenic-rich groundwater and other solutes are advancing progressively from the lacustrine margins toward the main granular aquifer, due to reversal of hydraulic gradients caused by intensive groundwater exploitation and the reduction in freshwater runoff provoked by dam construction on the main rivers. Desorption of arsenic will incorporate additional concentrations of arsenic into the groundwater and continue to have significant negative effects on human health and the environment.