We have analyzed the DNA sequences required for termination of Xenopus 5s RNA synthesis in vitro. Termination occurs within clusters of four or more consecutive T residues in the noncoding DNA strand sequence. The 3’flanking sequence following the gene is not required for termination. The distance between the T cluster and the site of transcription initiation as well as the exact nucleotide sequences preceding the T cluster can be varied without impairing termination. Thus the sequence signals for accurate initiation and termination of transcription are separable and can function independently. No significant dyad symmetry near the end of the gene appears to be required, in contrast to termination sites in procaryotes. However, the nucleotide sequences adjacent to the T cluster influence the efficiency of transcription termination. Efficient termination is observed whenever GC-rich sequences surround the T cluster. The inefficient termination of transcription of X. laevis major oocyte type 5s RNA genes appears to result from groups of two or more consecutive A residues within three nucleotides preceding or following the T cluster. Other variables, such as the temperature, the nucleotide concentration and the addition of (Y-amanitin, also influence the efficiency of termination.