Trimethylamine oxide (TMAO), which has previously not been known to occur in significant amounts in the blood of marine teleosts, rose to concentrations of approximately 50 mM in the blood of winter-acclimatized rainbow smelt, Osmerus mordax. Urea also increased in the blood of cold-acclimatized smelt, and, with TMAO, contributed significantly to the winter freezing point depression. TMAO and urea also varied seasonally in muscle and liver tissues. TMAO and urea appeared to be reabsorbed from the urine. Losses of TMAO and urea from the head region of the fish, where most of the losses appeared to occur, were approximately 9 μmol and 8 μmol 100 g⁻¹ h⁻¹, respectively. Despite the effluxes, TMAO and urea levels in both the blood and muscle either increased or were maintained in starved, cold-acclimated fish, suggesting that they were synthesized in response to cold temperature. TMAO was also found in the blood of some other cold-hardy teleosts.