An Early Pleistocene fluvio-lacustrine sequence from the extremely arid southern Negev desert, Israel, indicates that climatic conditions during that period were humid enough to support the formation of a lake (Lake Zihor) there. The lacustrine sediments were deposited in a tectonic valley that developed along the Zihor tectonic line after the deposition of the Pliocene Arava Formation. They intercalate with fluvial sediments that contain several horizons of reddish calcic paleosols. The lacustrine sequence comprises three sedimentary cycles. Each starts with a dark clay layer overlain by a white limestone unit, both of which contain abundant freshwater fauna, and terminates with a green detrital limestone that contains only one type of brackish-water ostracod. The top of each green limestone unit is characterized by pedogenic features and microrelief, indicating desiccation of the lake and soil development. Isotope data support the hypothesis that the white limestone was deposited in an open, freshwater lake. The incision of the present channel of Nahal (wadi) Zihor in the lacustrine sediments is manifested by a series of rock-cut and fluvial terraces (Q1–Q4) capped by gypsic-salic soils, which reflect the onset of the present, extremely arid climate. Over 100 find-spots and larger occurrences of prehistoric artifacts assigned to the Lower Paleolithic were discovered near Lake Zihor. On the basis of techno-typological and stratigraphic considerations, these assemblages are divided into two groups, the first of which may be contemporaneous with the lake, while the second is found mainly on the younger Q1 and Q2 terraces. It is estimated that the lake existed for more than 100,000 years. The climate during this period was probably semiarid, but water budget calculations suggest that, in addition to surface runoff, seepage of groundwater along the Zihor Line contributed a considerable amount of water to the lake.