Insertional mutagenesis based on maize Activator/Dissociator (Ac/Ds) transposons is becoming a major approach used to produce a saturated mutant collection in rice. In this research, Ds-T-DNA transformed homozygotes were crossed with Ac-T-DNA transformed homozygotes in order to establish an Ac/Ds transposon system in rice. The successive investigation of Ds transposition from F 1 to F 5 generations indicated that the frequencies of germinal transposition increased over successive generations and reached 54.2% in F 3 generation. The Ds transposition pattern revealed that a Ds transposition induced an approximately 170-bp deletion of T-DNA sequence and another Ds transposition carried a 272-bp T-DNA sequence. Using thermal asymmetric interlaced PCR (TAIL-PCR), some flanking sequences of the Ds element were amplified. Analyses of 17 Ds-flanking sequences showed that five Ds were inserted into gene regions. The Ds could transpose not only to the linked sites but also to the unlinked sites. The frequency of inter-chromosomal transposition of Ds was 33.3%.