A two-element Activator/Dissociation (Ac/Ds) gene trap system was successfully established in rice (Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds flanking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identified on chromosome 7 within a 40-kbp region. One-third of Ds flanking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), confirming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast artificial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the efficiency of this system is suitable for large-scale transposon mutagenesis in rice.