We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.