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Rainbow smelt (Osmerus mordax) tolerate temperatures close to the freezing point of sea water, in part, through the use of glycerol as an antifreeze. Potential mechanisms for glycerol production by liver were assessed by comparing activities of key enzym es of carbohydrate and amino acid metabolism in rainbow smelt to those in Atlantic tomcod (Microgadus tomcod) and smooth flounder (Liopsetta putmani). The latter two species inhabit the same environment but do not maintain high levels of blood glycerol. The enzyme profile of liver from rainbow smelt is substantially different from those of the other species and is poised for glycerol production. With respect to carbohydrate metabolism, glycerol-3-phosphate dehydrogenase activity in rainbow smelt liver was 156 µmoles min-1g-1, a level which was 28 and 12-fold higher than activities in tomcod and flounder liver, respectively. Glycerol-3-phosphatase activity in smelt liver was 1.95 µmol min-1g-1. This activity was 2.7 and 5.4-fold higher than those in tomcod and flounder liver, respectively. As such, the production of glycerol appears to be dependent upon the concerted action of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. The enzyme profile also suggests that amino acids are a potential source of carbon for glycerol. Aspartate aminotransferase activity in rainbow smelt was 7 to 14-fold higher in comparison to tomcod and flounder liver, respectively. Activities of alanine aminotransferase and glutamate dehydrogenase in liver were between 2 and 3-fold higher in rainbow smelt than in the other two species. Finally, it is shown that in vitro preparations of smelt liver sections produce glycerol at 0°C lending support to the concept that liver is a site of glycerol synthesis in vivo.