Viral-based techniques are the most efficient systems to deliver DNA into stem cells because they show high gene transduction and transgene expression in many cellular models. However, the use of viral vectors has several disadvantages mainly involving safety risks. Conversely, nonviral methods are rather inefficient for most primary cells. The Nucleofector technology, a new nonviral electroporation-based gene transfer technique, has proved to be an efficient tool for transfecting hard-to-transfect cell lines and primary cells. However, little is known about the capacity of this technique to transfect adult stem cells. In this study, we applied the Nucleofector technology to engineer human bone marrow- derived mesenchymal stem cells (hMSCs). Using a green fluorescent protein reporter vector, we demonstrated a high transgene expression level using U-23 and C-17 pulsing programs: 73.7%+/-2.9% and 42.5%+/-3.4%, respectively. Cell recoveries and viabilities were 38.7%+/-2.9%, 44.5%+/-3.9% and 91.4%+/-1.3%, 94.31%+/-0.9% for U-23 and C-17, respectively. Overall, the transfection efficiencies were 27.4%+/-2.9% (U-23) and 16.6%+/-1.4% (C-17) compared with 3.6%+/-2.4% and 5.4%+/-3.4% of other nonviral transfection systems, such as FUGENE6 and DOTAP, respectively (p<.005 for all comparisons). Nucleofection did not affect the immunophenotype of hM-SCs, their normal differentiation potential, or ability to inhibit T-cell alloreactivity. Moreover, the interleukin-12 gene could be successfully transfected into hMSCs, and the immunomodulatory cytokine was produced in great amount for at least 3 weeks without impairment of its biological activity. In conclusion, nucleofection is an efficient nonviral transfection technique for hMSCs, which then may be used as cellular vehicles for the delivery of biological agents.