Tetrodotoxin-resistant Nav1.5 Na⁺ channel has been considered as the cardiac sodium channel. Na⁺ currents with tetrodotoxin resistance (TTX-R) and Nav1.5/SCN5A mRNA have been observed in neurons, but the cDNA encoding the TTX-R Nav1.5 Na⁺ channels in human central nervous system (CNS) has not been identified. Nav1.5/SCN5A cDNA was first cloned from human brain cortex by using RT-PCR method. Two variants of Nav1.5/SCN5A were found and tentatively named hB1 and hB2.Full sequence of cDNA encoding the α-subunit of TTX-R Nav1.5 Na⁺ channel in human brain cortex was 6 201 nucleotides long and was designated hB1. The longest open reading frame of hB1 (accession number EF629346 ) encodes 2 016 amino acid residues. Sequence analysis has indicated that hB1 is highly homologus with human cardiac Nav1.5/SCN5A with >98% amino acid identity. There are 28 different amino acids between them, with 7 of which locating in the region encoded by exon6A or exon6. Alternative splicing of exon18 was not found in the gene cloning of human brain Nav1.5/SCN5A, which was different from human heart Nav1.5/SCN5A, but a novel alternative splicing lacking exon24 was first found. The two variants were detected in similar ratio in brain, but they were proved to relate to age development in heart tissue. The exon24 of human Nav1.5/SCN5A has 54 nucleotides, encoding 30 amino acid residues, and are located in human chromosome 3P21. This alternative splicing was also found in other tissues other than heart and brain. The expression pattern of the two variants in different tissues was different when detected by competitive PCR method and it was also changing with age development. Furthermore,Nav1.5/SCN5A mRNA was detected in 16 different tissue types of Wistar rats (P80) by reverse polymerase chain reaction (RT-PCR). These results suggest that Nav1.5 Na⁺ channels in human brain are encoded by new variants of Nav1.5/SCN5A and its mRAN is more widely expressed than previously thought. The study is useful for making further investigation in the functional analysis of Nav1.5 Na⁺ channels in different tissues.