TIt has previously been reported that transcription In vivo of the tRNASec gene requires three promoter elements, a PSE and a TATA-box upstream of the coding region which are functionally interchangeable with the U6 snRNA gene counterparts and an Internal B-block, resembling that of classical tRNA genes (1). We have established an In vitro transcription system from HeLa cells in which three factors, which are either essential for or stimulate transcription were identified. Apart from the TATA-blnding protein TBP, the PSE-blnding protein PBP was found to be essentially required for expression of the gene. Depletion of PBP from cell extracts by PSE-oligonucleotides abolished tRNASec transcription, which could be reconstituted by re-addition of partially purified PBP. Addition of Increasing amounts of recombinant human TBP to an S100 extract stimulated transcription of the tRNASec, the mouse U6 snRNA and the human Y3 genes, an effect which was not observed In the case of a TATA-less tRNA gene. Purified human TFIIA strongly stimulated tRNASec transcription in a fashion depending on the concentration of TBP. Surprisingly, partially purified TFIIIC was shown to be dispensable for transcription In vitro and unable to bind the B-block of this gene In vitro, although its sequence matches the consensus for this element. Collectively, these data suggest that the mechanism by which transcription complexes are formed on the tRNASec gene is dramatically different from that observed for classical tRNA genes and much more resembles that observed for externally controlled pol III genes.