We describe a cell-free transcription system for the archaebacterium Sulfolobus sp. B12 that specifically initiates transcription at the 5S rRNA-encoding DNA and the 16S/23S rRNA-encoding DNA promoters of the same species. With this crude extract system, specific initiation was absolutely dependent on the box A motif, a highly conserved promoter element in archaebacteria located approximately 25 base pairs upstream of transcription initiation sites. In vitro transcription of the rRNA genes by purified RNA polymerase, however, resulted in semi-specific, box A-independent initiation, indicating that factor(s) in the crude extract were necessary for the highly specific box A-dependent transcription. Fractionation of the cell-free extract by sucrose-gradient centrifugation resulted in the identification of a low molecular weight fraction complementing purified RNA polymerase to an extract-like specificity.