Summary Microorganisms can utilize different sugars as energy and carbon sources and the genes involved in sugar metabolism often exhibit highly regulated expression. To study cis-acting elements controlling arabinose-responsive expression in archaea, the promoter of the Sulfolobus solfataricus araS gene encoding an arabinose binding protein was characterized using an Sulfolobus islandicus reporter gene system. The minimal active araS promoter (P(araS)) was found to be 59 nucleotides long and harboured four promoter elements: an ara-box, an upstream transcription factor B-responsive element (BRE), a TATA-box and a proximal promoter element, each of which contained important nucleotides that either greatly decreased or completely abolished promoter activity upon mutagenesis. The basal araS promoter was virtually inactive due to intrinsically weak BRE element, and the upstream activating sequence (UAS) ara-box activated the basal promoter by recruiting transcription factor B to its BRE. While this UAS ensured a general expression from an inactive or weak basal promoter in the presence of other tested carbon resources, it exhibited a strong arabinose-responsive transcriptional activation. To our knowledge, this represents the first example of an archaeal UAS that exhibits differential activation to the expression on the same promoter in the presence of different carbon sources.