Genome and expressed sequence tag sequencing in rice provides a vast resource of gene sequences whose functions need to be determined by reverse genetics methods for expression and mutational analysis. To develop insertional mutagenesis strategies in rice, we transformed japonica and indica cultivars with maize transposon constructs, for knockout and gene detection insertions. A green fluorescent protein (GFP) excision assay developed enabled the visualization of transposon excision in a variety of tissues. Surprisingly, early Ac excision was observed directly after transformation from a construct containing the strong double CaMV enhancer element adjacent to the Ac promoter. We identified genotypes with Ac amplification events and with a forward transposition rate of 15-50% that are useful for generating lines containing multiple transposons. The sequence of DNA flanking transposed Ac provided a resource of Ac-tagged sites, which represented about 50% in the target region, indicating insertional specificity appropriate for the identification of mutants of sequenced genes. Clustered Ac transposition was revealed by six insertions in 70 kb of chromosome 6. Gene detection Ac-Ds enhancer trap and activation tag transformants revealed active transposition in about half the lines. These resources for functional genomics are developed by an EU-funded consortium and will be made available to rice researchers worldwide.