A first-line gene therapy for type 1 diabetes should be based on a safe procedure to engineer an accessible tissue for insulin release. We evaluated the ability of the skeletal muscle to release human insulin after electrotransfer (ET)-enhanced plasmid DNA injection in mice. A furin-cleavable proinsulin cDNA under the CMV or the MFG promoter was electrotransferred to immune-incompetent mice with STZ-induced severe diabetes. At 1 week, mature human insulin was detected in the serum of 17/20 mice. After an initial peak of 68.5 +/- 34.9 microU/ml, insulin was consistently detected at significant levels up to 6 weeks after gene transfer. Importantly, untreated diabetic animals died within 3 weeks after STZ, whereas treated mice survived up to 10 weeks. Fed blood glucose (BG) was reduced in correspondence with the insulin peak. Fasting BG was near-normalized when insulin levels were 12.9 +/- 5.3 (CMV group, 2 weeks) and 7.7 +/- 2.6 microU/ml (MFG group, 4 weeks), without frank hypoglycemia. These data indicate that ET-enhanced DNA injection in muscle leads to the release of biologically active insulin, with restoration of basal insulin levels, and lowering of fasting BG with increased survival in severe diabetes. Therefore the skeletal muscle can be considered as a platform for basal insulin secretion.