RNA polymerase I terminates transcription of mouse rDNA 565 bp downstream of the 3' end of mature 28S rRNA. This specific termination event can be duplicated in a nuclear extract system. RNA molecules with authentic 3' ends are transcribed from ribosomal minigene constructs provided the templates retain a minimal length of downstream spacer sequences. The nucleotide sequence of the region of transcription termination contains a set of repetitive structural elements consisting of 18 bp conserved nucleotides surrounded by stretches of pyrimidines. Termination in vivo occurs within the first element. This site is preferentially used in vitro at low template concentrations. At increasing DNA concentrations a termination site within the second repetitive element is used. Competition experiments with defined 3'-terminal fragments suggest that transcription termination by RNA polymerase I requires interaction of some factor (or factors) with the repetitive structural elements in the 3' nontranscribed spacer.