A control region that directs accurate initiation of 5s RNA synthesis is located within the middle third of the X. borealis somatic 5S RNA gene. The 5’boundary of this region is described in the accompanying paper (Sakonju, Bogenhagen and Brown, 1980) as occurring between residues 50 and 55 of the 120 nucleotide 5S RNA gene. In this paper we delimit the 3’boundary of this control region between gene residues 80 and 83. The 3’boundary has been defined by in vitro transcription of a series of deletions of cloned 5S DNA whose endpoints approach and enter the gene from the 3’side. Since these deletions lack the normal termination site of 5S RNA synthesis, we have developed an assay for accurate initiation of transcription in the absence of correct termination. In vitro transcription in the presence of cordycepin triphosphate(3’(IATP) causes synthesis of a reproducible array of shortened RNA molecules displayed by polyacrylamide gel electrophoresis. This transcription assay shows that 3’deletions leaving the first 83 or more 5’gene residues support accurate initiation of transcription. Larger deletions leaving 80 or fewer gene residues do not support 5S RNA-specific transcription initiation. The gene region from nucleotides 41-87 which contains this control region has been cloned without any additional 5S DNA sequences. This internal gene fragment contains sufficient information to direct specific transcription initiation at a site in the upstream plasmid sequence.