The scarcity of C4 plants in cool climates is usuallyattributed to their lower photosynthetic efficiency thanC3 species at low temperatures. However, a lowerfreezing resistance may also decrease the competitiveadvantage of C4 plants by reducing canopy duration,especially in continental steppe grasslands, wherea short, hot growing season is bracketed by frostevents. This paper reports an experimental test of thehypothesis that cold acclimation is negligible inC4 grasses, leading to greater frost damage than in C3species. The experiments exposed six C3 and three C4Mongolian steppe grasses to 20 d chilling or controlpre-treatments, followed by a high-light freezing event.Leaf resistance to freezing injury was independent ofphotosynthetic type. Three C3 species showed constitutivefreezing resistance characterized by <20% leafmortality, associated with high photosynthetic carbonfixation and electron transport rates and low leafosmotic potential. One freezing-sensitive C4 speciesshowed the expected pattern of chilling-induced damageto photosynthesis and >95% leaf mortality afterthe freezing event. However, three C3 and two C4species displayed a cold acclimation response, showingsignificant decreases in osmotic potential andphotosynthesis after exposure to chilling, and a30–72% reduction of leaf freezing injury. This resultsuggested that down-regulation of osmotic potentialmay be involved in the cold acclimation process, anddemonstrated that there is no inherent barrier to thedevelopment of cold acclimation in C4 species fromthis ecosystem. Cold acclimation via osmoregulationrepresents a previously undescribed mechanism toexplain the persistence of C4 plants in cool climates.