The effect of thiamine deficiency on insulin biosynthesis was studied. In thiamine deficient rats the total pancreatic protein content was not altered when compared to control rats whereas the pancreatic insulin content was decreased. Though the in vivo incorporation of 3H-leucine and the in vivo conversion of U-14C-glucose into proinsulin and insulin were not affected in thiamine deficient rats, the tolbutamide induced increased in vivo incorporation of 3H-leucine and in vivo conversion of U-14C-glucose into proinsulin and insulin was not seen in thiamine deficient rats. These results suggest that the biosynthesis of insulin is impaired in thiamine deficiency. Even tolbutamide could not increase the biosynthesis of insulin in this condition.