To achieve high transfection efficiency in human fibroblasts with good preservation of proliferative capacity we developed an electroporation procedure that combines two distinct modalities: use of recipient cells synchronized in the late G2/mitotic phase of the cell cycle and treatment of cells post-electroporation with 5 mM butyrate. This combination enabled reduction of plasmid DNA concentration and electroporation voltage, both associated with cytotoxicity, while greatly enhancing transfection efficiencies. Although the method was primarily developed for transient expression it was also found to improve stable expression. This procedure should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.