Transcription of the S14 gene in primary hepatocytes is stimulated in response to increased carbohydrate metabolism. We have demonstrated previously that a 30-base pair (bp) segment of the S14 gene from -1457 to -1428 is a carbohydrate response element (ChoRE). This element contains a (5')CACGTG motif that is essential for control. DNase I footprinting experiments with liver nuclear extract revealed two factors binding within the S14 ChoRE. In transient transfection experiments, mutation of the upstream site between -1457 and -1450 did not affect the response to elevated glucose, whereas the downstream 21-bp site between -1448 and -1428 was sufficient to mediate the glucose induction. Electrophoretic mobility shift assays indicated that the hepatic factor binding to this site in vitro is closely related or identical to the major late transcription factor (MLTF). However, replacement of the 21-bp S14 ChoRE with the authentic MLTF binding site from the adenovirus major late promoter failed to elicit the glucose response. By systematically exchanging bases between the functional S14 and nonresponsive adenovirus sites, the sequence (5')CACGTGNNNGCC was found to be essential for carbohydrate regulation. A segment containing this specific motif from the rat fatty acid synthase gene, another carbohydrate-responsive gene in hepatocytes, conferred a carbohydrate response when linked to the S14 promoter. Thus, the context of the CACGTG motif provides the specificity for regulation by carbohydrate metabolism.