Studies on formation of the transcription initiation complex on the tRNA(Tyr) gene in yeast extracts with the use of Sarkosyl-challenge assay proved that a single magnesium ion is indispensable for the complex assembly in the absence of nucleotides. Other cations do not substitute for Mg2+. The optimal KC1 concentration for transcription of the gene by the assembled initiation complex was found to be twice as high as that for the assembly process and close to that observed previously for the TFIIIC (or tau) factor dissociation from the promoter. The preformed complex remained stable for several hours at room temperature and its decay was not influenced by ionic strength. The data seem to support the notion that the TFIIIC factor is used only for assembly of the initiation complex and is not necessarily involved in the subsequent steps of transcription.