Background Mesenchymal stem cells (MSC) are widely regarded as a promising tool for cellular therapy applications, and genetic modification by safe, liposome-based vectors may enhance their therapeutic potential.Methods The present study describes the use of a cationic lipid vector (Lipofectamine 2000 (TM)) to deliver genes to MSC isolated from a number of species in vitro and determined the characteristics of this vector system in terms of dose, toxicity and the time course of expression. In addition, the optimal use of a nuclear localization sequence (NLS) to enhance gene expression was explored.Results Lipofection of human MSC did not adversely affect their ability to differentiate into osteogenic- and adipogenic lineages. Although human and rat MSC were found to take up lipoplexes with relative efficiency, lower levels of gene expression were detected in rabbit MSC, demonstrating a crucial effect of species. Peptides containing reiterated motifs of NLS were found to significantly improve on the level of transgene expression. Optimal gene delivery was observed when a three-fold reiterated NLS sequence was included in the liposome formulation.Conclusions Thus, nonviral gene delivery to MSC is feasible with efficiency being species dependent and can be enhanced by use of a three-fold reiterated NLS. Copyright (C) 2010 John Wiley & Sons, Ltd.