Transformation of the well-studied maize transposable elements into other plant species should enable transposon tagging methodology to be used for the isolation of interesting genes in the heterologous host. Here we describe the isolation of a transposon-tagged male sterile mutant in Arabidopsis thaliana using the maize Enhancer-Inhibitor transposable element system introduced into Arabidopsis. The mutant lacks pollen, preventing normal self-fertilization, a characteristic important for production of hybrid seed in many crop plants. We have identified an Enhancer-transposase-mediated Inhibitor element insertion responsible for the male sterile phenotype, and isolated the corresponding gene named MALE STERILITY 2. Critical evidence that the Inhibitor-element-containing gene is involved in the male sterile phenotype is provided by the DNA sequences of new excision-derived alleles from independent stable fertile and male sterile progeny of the original mutant.