Four or more consecutive thymidine residues on the non-template strand and G + C-richness of flanking DNA are the two necessary characteristics of efficient RNA polymerase-III-dependent transcriptional terminators. We have identified, from the study of in vitro transcription of a human Alu family repeat, a new type of RNA polymerase-III-dependent transcriptional terminator. A 258 base-pair Alu repeat located on the 3' side of the human alpha 1 globin gene can be transcribed in a HeLa S-100 extract to generate three RNA species of lengths 404 to 408, 252 to 255 and 173 to 174 nucleotides, respectively. Kinetics, pulse-chase and RNA incubation experiments showed no significant internal processing of the longer transcripts into shorter ones. These data plus detailed RNA mapping demonstrated conclusively that the multiple Alu RNA species resulted from accurate initiation at the first base (5' end) of the repeat, and multiple termination downstream. The 3' end(s) of the major transcript (252 to 255 nucleotides) maps at the 3' end of the Alu repeat sequence where there are not four or more consecutive thymidine residues on the non-template strand. The functional domain of the terminator has been mapped to a 45 base-pair segment that includes 36 base-pairs of the 3' end sequence of the Alu repeat plus nine base-pairs downstream. The high efficiency of termination (greater than 90%), the lack of consecutive T residues, the richness in A + T content, and the proposed ability of the RNA to form an imperfect hairpin structure in the 3' region of the transcript, thus identify a new type of eukaryotic class III terminator. We compare the structure of this class III terminator with that of the bacterial rho-dependent terminator. We also discuss its implication in the mechanism(s) of amplification and dispersion of Alu sequences in the primate genomes.