Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed.