The purpose of these studies was to determine the time course for onset of effects of hypoglycemia on insulin gene transcription in vivo. Using insulin infusions, we found that insulin-induced hypoglycemia decreased levels of precursors for insulin mRNA, reflecting changes in new mRNA synthesis, to new steady-state values within 100 min. These changes were followed by declines in processed insulin mRNA. An alternate infusion technique was developed to lower plasma glucose levels from a constant level of 120-130 to 50-60 mg/dl in <10 min without changing insulin levels from those maintained during a preceding 1-h control period. Using this protocol, we found that levels of precursors for insulin mRNA remained constant for the first 20 min of hypoglycemia, then decreased rapidly at 40 and 60 min. The initial delay followed by rapid decline suggests that the decrease of insulin gene transcription in response to hypoglycemia is an active process requiring one or more inductive events before implementation.