Expression of the mouse pancreatic amylase gene Amy-2.2 is increased approximately 10-fold in response to increasing the carbohydrate content of the diet from 9.6 to 74%. The DNA sequence mediating this response has been localized to the 5' flanking region of the amylase gene by analysis of hybrid constructs in transgenic mice. The results define a 127-base pair dietary response unit that includes two previously described regulatory elements, an insulin-responsive element and a pancreatic enhancer. Fragments containing these two elements alone fail to respond to diet, demonstrating a requirement for additional regulatory sequences. Another mouse amylase gene Amy-2.1 is only minimally responsive to insulin and to diet. The data are consistent with the hypothesis that the insulin-response element is necessary but not sufficient for regulation of amylase by dietary carbohydrate.