We have performed in vitro kinetic analyses of transcription from the 5S gene promoter in order to resolve the rate-limiting events which lead to accurate transcription of the 5S RNA gene. We demonstrate that a rate-limiting intermediate can be formed during an extended incubation prior to initiation of transcription. Formation of such a complex is temperature-dependent, requires magnesium and ATP, consists of stoichiometric amounts of the known class III transcription factors and RNA polymerase III on 5S DNA, and eliminates the normal lag in attainment of a steady-state rate of transcription. This complex is therefore different from the "stable complex" minimally required for template commitment. Further analyses demonstrate that TFIIIB, like TFIIIA and TFIIIC, can be stably sequestered on the 5S gene and also allow us to formulate the following order of factor interactions on the 5S gene: TFIIIA, TFIIIC, TFIIIB, RNA polymerase III.