A new type of termination signal for RNA polymerase III transcription has been identified. A cloned DNA from salmon highly repetitive sequences, designated as Sm2, serves as a template for an RNA with about 140 nucleotides, but the clone does not have four or more consecutive T residues in the presumed termination site. S1 nuclease mapping analysis clearly demonstrated that termination occurred at the beginning of the AT-rich sequence in the 3' region of Sm2. Studies with a series of 3'-deletion mutants strongly suggested that an AT sequence of more than nine nucleotides functioned as a terminator. Furthermore, results with another series of 3'-deletion mutants showed that a sequence of only nine nucleotides (ATATATATT) in the noncoding strand in fact functioned as a terminator (designated as the 9AT terminator), although with relatively low efficiency compared to a tract of TTTT introduced downstream, and that new cryptic termination sites were introduced in the upstream slight AT-rich region by approach of the 9AT terminator. An AT-rich region downstream from the actual termination site of Sm2 DNA may enhance the efficiency of termination by facilitating detachment of RNA polymerase from the template DNA. Since the nucleotide sequence around the termination site appears to be conserved in the salmon repetitive sequences, this new terminator may be responsible for the generation of a discrete-sized RNA transcribed from salmon total genomic DNA in vitro.