The elastase I gene enhancer that specifies high levels of pancreatic transcription comprises three functional elements (A, B, and C). When assayed individually in transgenic mice, homomultimers of A are acinar cell specific, those of B are islet specific, and those of C are inactive. To determine how the elements interact in the elastase I enhancer and to investigate further the role of the C element, we have examined the activity of the three possible combinations of synthetic double elements in transgenic animals. Combining the A and B elements reconstitutes the exocrine plus endocrine specificity of the intact enhancer with an increased activity in acinar cells compared with that in the A homomultimer. The B element therefore plays a dual role: in islet cells it is capable of activating transcription, whereas in acinar cells it is inactive alone but greatly augments the activity specified by the A element. The C element augments the activity of either the A or B element without affecting their pancreatic cell type specificity. The roles of each element were verified by examining the effects of mutational inactivation of each element within the context of the elastase I enhancer. These results demonstrated that when tested in animals, the individual enhancer elements can perform discrete, separable functions that combine additively for cell type specificity and cooperatively for the overall strength of a multielement stage- and site-specific transcriptional enhancer.