Voltage‐gated sodium (Na⁺) channels typically contain a pore‐forming α subunit and one or two auxiliary β subunits. Although initial characterization of known α and β subunits has been facilitated by expression in heterologous cells, to understand fully the differences between individual subunits and the functional consequences of selective subunit expression, there is a need to acutely manipulate expression in cells that endogenously express Na⁺ channels. To this end, we have constructed a recombinant adenovirus containing a cDNA for a mouse Na⁺ channel β1 subunit with a yellow fluorescent protein fused to its C‐terminus (Ad‐β1‐EYFP), and with fluorescence microscopy detected β1‐EYFP expression in primary cerebellar neurons and Chinese hamster ovary (CHO) cells upon transduction with this adenovirus, including expression in the plasma membrane. Consistent with this, patch clamp recordings confirmed that Na⁺ currents in CHO cells expressing mouse Na_v1.4 α subunits were appropriately modified by the viral‐mediated expression of β1‐EYFP subunits. The results demonstrate that adenoviral‐mediated gene delivery can be used effectively to express epitope‐tagged Na⁺ channel subunits with properties similar to wild‐type subunits, and suggest that Ad‐β1‐EYFP will be a useful reagent for investigating Na⁺ channels in a variety of excitable cell types, including neurons. © 2003 Wiley‐Liss, Inc.