Transfection experiments using Xenopus vitellogenin A2 gene constructs allowed us to identify an activator which increases the activity of the thymidine kinase promoter. The activator is located between -121 and -87 of the A2 vitellogenin gene and is separated by a stretch of curved DNA from the estrogen-responsive DNA element at -331. The activator functions in a cell-specific manner, as it is active in human breast cancer cells (MCF-7) as well as hepatoma cells but not in fibroblasts or HeLa cells. The activator is composed of at least three elements: elements 1 and 2 which form a partial palindrome, function independently, but act synergistically when combined. Element 3 is not active on its own, but supports elements 1 and 2. A TATA box region derived from the Xenopus albumin gene is sufficient for the function of the activator. In vitro transcription experiments using rat liver nuclear extracts demonstrate that the activator interacts with transcription factors. These factors are distinct from those recognizing HP1, a regulatory element common to several genes specifically expressed in hepatocytes.