We investigated the ecological physiology and behavior of free-living wood frogs [Lithobates (Rana) sylvaticus] overwintering in Interior Alaska by tracking animals into natural hibernacula, recording microclimate, and determining frog survival in spring. We measured cryoprotectant (glucose) concentrations and identified the presence of antifreeze glycolipids in tissues from subsamples of naturally freezing frogs. We also recorded the behavior of wood frogs preparing to freeze in artificial hibernacula, and tissue glucose concentrations in captive wood frogs frozen in the laboratory to −2.5°C. Wood frogs in natural hibernacula remained frozen for 193±11 consecutive days and experienced average (October-May) temperatures of −6.3°C and average minimum temperatures of -14.6±2.8°C (range −8.9 to −18.1°C) with 100% survival (N=18). Mean glucose concentrations were 13-fold higher in muscle, 10-fold higher in heart and 3.3-fold higher in liver in naturally freezing compared with laboratory frozen frogs. Antifreeze glycolipid was present in extracts from muscle and internal organs, but not skin, of frozen frogs. Wood frogs in Interior Alaska survive freezing to extreme limits and durations compared with those described in animals collected in southern Canada or the Midwestern United States. We hypothesize that this enhancement of freeze tolerance in Alaskan wood frogs is due to higher cryoprotectant levels that are produced by repeated freezing and thawing cycles experienced under natural conditions during early autumn.