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Lake Petén Itzá (Guatemala) possesses one of the longest lacustrine sediment records in the northern Neotropics, which enabled study of paleoclimate variability in the region during the last ∼400 000 years. We used geochemical (Ti, Ca/(Ti+Fe) and Mn/Fe) and mineralogical (carbonates, gypsum, quartz, clay) data from sediment core PI-2 to infer past changes in runoff, lake evaporation, organic matter sources and redox conditions in the water column, caused by hydrological changes in the northern Neotropics during Marine Isotope Stages (MISs) 3–2. From 59 to 39 cal ka BP climate conditions were relatively wet, and the lake was marked by higher primary productivity and anoxic bottom waters. This wet environment was interrupted for two periods of possible low water level at 52 and 46 cal ka BP, when our data suggest higher evaporation, high terrestrial organic matter input and persistent oxic conditions. Between 39 and 23 cal ka BP, evaporation and input of terrestrial organic matter increased considerably, lake level declined, and lake bottom waters generally became oxic. These conditions reversed during the Last Glacial Maximum (23.5–18.0 cal ka BP), when runoff and lake productivity increased, and rising lake level caused bottom waters to again become anoxic. Comparison of our hydrologic proxy data with sea surface temperature anomalies between the eastern Pacific and the Caribbean suggests that changes in the intensity of the Caribbean Low-Level Jet (CLLJ) may have influenced long-term changes in runoff during MISs 3–2. Higher intensity of the CLLJ during the onset of MIS 3 and the LGM might have led to greater runoff into the lake, whereas the MIS 3–2 transition experienced a weaker CLLJ and consequently less runoff. A refined, high-resolution age–depth model for the PI-2 sediment core enabled us to identify millennial-scale Greenland interstadials (GIs) 14–2, Greenland stadials (GSs) 14–2 and Heinrich stadials (HSs) 5–1. In general, HSs and GSs were characterized by drier conditions. In contrast to GSs and HSs, GIs were characterized by greater runoff and overall wetter conditions, with the most pronounced GI peaks between 40 and 30 cal ka BP. Whereas GSs 9, 8, 7 and 6 began with abrupt increases in evaporation and ended with gradual increases in humidity, GSs 11 and 10 showed reversed patterns. The Lake Petén Itzá paleohydrology record, along with other regional paleoclimate records, led us to conclude that shifts in the position of the Intertropical Convergence Zone (ITCZ) altered moisture delivery to the lake on millennial timescales. During GSs and HSs, high evaporation from Petén Itzá (dry climate conditions) was associated with a more southerly position of the ITCZ, whereas wetter GIs prevailed during a more northerly ITCZ position. Although abrupt millennial-scale shifts in ITCZ and hydroclimate between GSs/HSs and GIs can be linked to instabilities in the Atlantic Meridional Overturning Circulation (AMOC), longer-term changes were additionally influenced by changes in atmospheric convection linked to modulations of the CLLJ in response to ΔSST between the equatorial Pacific and tropical Atlantic.