Tetrodotoxin-resistant Na+currents are expressed in a variety of muscle cells including human jejunal circular smooth muscle (HJCSM) cells. The aim of this study was to determine the molecular identity of the pore-forming alpha-subunit of the HJCSM Na+ channel. Degenerate primers identified a cDNA fragment of 1.5 kb with 99% nucleotide homology with human cardiac SCN5A. The identified clone was also amplified from single smooth muscle cells by reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed expression of full-length SCN5A. Laser capture microdissection was used to obtain highly purified populations of HJCSM cells. RT-PCR on the harvested cells showed that SCN5A was present in circular but not in longitudinal muscle. A similar result was obtained using a pan-Na+ channel antibody. The full-length sequence for SCN5A was obtained by combining standard polymerase chain reaction with 5' and 3' rapid amplification of cDNA end techniques. The intestinal SCN5A was nearly identical to the cardiac SCN5A. The data indicate that SCN5A is more widely distributed than previously thought and encodes the pore-forming alpha-subunit of the tetrodotoxin-resistant Na+ current in HJCSM cells.