The nucleotide sequence of the whole mini-Rts1 genome consisting of 1,855 base pairs was determined. In addition to the cluster of five 24-base-pair direct repeats previously detected (Y. Kamio and Y. Terawaki, J. Bacteriol. 155:1185-1191, 1983), another cluster of three 21-base-pair direct repeats was found. All eight repeats were located in one direction and contained a consensus sequence TTCCCCPyPyPuPuCACACACC. Between the two clusters, a large open reading frame that could encode a 32,980-dalton polypeptide consisting of 288 amino acids was assigned. The molecular size predicted from the amino acid composition was close to the value of a unique mini-Rts1 product, the RepA protein, newly determined in minicells. A copy mutant of mini-Rts1 obtained in vitro by hydroxylamine treatment contained two-base-pair substitutions, one of which was located in the RepA protein coding region, and the other was close to the region where the oriC homologous sequences exist.