The feasibility of using nonintegrating lentiviral vectors has been demonstrated by recent studies showing their ability to maintain transgene expression both in vitro and in vivo. Furthermore, human immunodeficiency virus-1 (HIV-1) vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date, a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. In this study, we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector's U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained.