Maize Activator (Ac) is one of the prototype transposable elements of the hAT transposon superfamily, members of which were identified in plants, fungi, and animals. The autonomous Ac and nonautonomous Dissociation (Ds) elements are mobilized by the single transposase protein encoded by Ac. To date Ac/Ds transposons were shown to be functional in approximately 20 plant species and have become the most widely used transposable elements for gene tagging and functional genomics approaches in plants. In this chapter we review the biology, regulation, and transposition mechanism of Ac/Ds elements in maize and heterologous plants. We discuss the parameters that are known to influence the functionality and transposition efficiency of Ac/Ds transposons and need to be considered when designing Ac transposase expression constructs and Ds elements for application in heterologous plant species.