Hydrodynamic administration of naked DNA was investigated as a method for in vivo expression of variant proteins involved in metabolic diseases, using short-chain acyl-CoA dehydrogenase (SCAD) deficient mice (BALB/cByJ) as a model. Human SCAD wild-type (WT) and two disease-associated SCAD variant proteins (R147W and G185S) were expressed in mouse liver by means of single injections of SCAD cDNA under the control of a ubiquitin promoter. SCAD expression was detected two days after injection. The activity decreased after the first week but continued to be detectable for at least 31 days after injection. Analysis of SCAD WT, R147W, and G185S proteins in liver cells showed that all three SCAD proteins were processed to the mature protein in mitochondria. Concomitantly, the SCAD activity in BALB/cByJ mice injected with SCAD WT, G185S, and R147W cDNA was 30, 39, and 13%, respectively, of the level in normal mice. A tendency to a reduction in the level of butyrylcarnitine in blood was observed although only approximately 5% of the liver cells expressed the SCAD protein. Thus, hydrodynamic gene transfer allows for functional testing of SCAD variant proteins in vivo.