In adipose tissue, the microsomal enzyme 1,2-acyl CoA:diacylglyceroltransferase-1 (DGAT1) plays an important role in triglyceride storage. Because DGAT1 is expressed in skeletal muscle as well, we aimed to directly test the effect of DGAT1 on muscular triglyceride storage by overexpressing DGAT1 using in vivo DNA electroporation. A pcDNA3.1-DGAT1 construct in saline was injected in the left tibialis anterior muscle of rats, followed by the application of eight transcutaneous pulses, using the contralateral leg as sham-electroporated control. Electroporation of the DGAT1 construct led to significant overexpression of the DGAT1 protein. The functionality of DGAT1 overexpression is underscored by the pronounced diet-responsive increase in intramyocellular lipid (IMCL) storage. In chow-fed rats, DGAT1-positive myocytes showed significantly higher IMCL content compared with the control leg, which was almost devoid of IMCL (1.99 +/- 1.13% vs. 0.017 +/- 0.014% of total area fraction; P <0.05). High-fat feeding increased IMCL levels in both DGAT1-positive and control myocytes, resulting in very high IMCL levels in DGAT1-overexpressing myocytes (4.96 +/- 1.47% vs. 0.80 +/- 0.14%; P <0.05). Our findings indicate that DGAT1 contributes to the storage of IMCL and that in vivo DNA electroporation is a promising tool to examine the functional consequences of altered gene expression in mature skeletal muscle.