We have introduced a genetically marked Dissociation transposable element (Dsneo) into tomato. In the presence of Ac transposase, Dsneo excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Dsneo (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Dsneo, we examined the genomic distribution of Dsneo receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Dsneo elements was conducted in two tomato families, derived from independent primary transformants each bearing Dsneo within a T-DNA at a unique position in the genome. Transposition of Dsneo generated clusters of insertions that were positioned on several different tomato chromosomes. Dsneo insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Dsneo transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Dsneo. We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Dsneo, is an effective tool for transposon tagging experiments in tomato.