The wood frog (Rana sylvatica) is a terrestrial hibernator that can accumulate urea as an osmoprotectant in autumn and winter. This study tested the hypothesis that elevated urea can also function as a cryoprotectant in this freeze-tolerant species. Performance characteristics (threshold stimulus voltage, maximal isometric twitch and tetanic contraction forces, and (1/2) fatigue time) of isolated gastrocnemius muscles were measured before and after experimental freezing at -1.5 degrees C for 18 h, followed by thawing. Frozen/thawed muscles exhibited reduced function relative to baseline (prefreeze) levels; however, muscles preincubated in a saline solution containing urea (80 mmol l(-1)) performed substantially better in some tests than muscles incubated without urea. Concentrations of urea in these treated muscles, approximately 65 mmol l(-1), were within the physiological range in winter R. sylvatica. Reducing tissue urea levels to approximately 33 mmol l(-1) resulted in a similar pattern of response, although the differences between urea-incubated and saline-incubated muscles were not statistically significant. Tests of cryoprotective efficacy were also performed on gastrocnemius muscles from R. pipiens, a closely related, but freeze-intolerant species that hibernates aquatically and thus has little need to accumulate urea. Urea-treated muscles from this species performed no better than muscles incubated in saline, attesting that freeze tolerance cannot be conferred simply by augmenting cryoprotectant levels. Overall, these results bolster an earlier report that urea accumulated in response to low moisture availability can serve a cryoprotective role in freeze-tolerant ectotherms.