A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.