Overlapping cDNA clones spanning the entire coding region of a Na channel were isolated from newt retina. The coding region predicts a 2,007 amino acid protein, designated nRNaCh (newt retina sodium channel), which is homologous to other Na channels. In situ hybridization indicated that nRNaCh is expressed exclusively in spiking neurons, where a tetrodotoxin (TTX)-resistant Na current has been recorded. Therefore, nRNaCh cDNA is sure to encode the TTX-resistant Na channel of newt retina. Sequence comparisons show that nRNaCh is more homologous to TTX-sensitive Na channels than to TTX-resistant Na channels. The length of the S5-S6 loop of repeat I of nRNaCh is similar to that of TTX-sensitive channels, whereas TTX-resistant Na channels have a deletion. The 3^rd position in the SS2 region of repeat I of nRNaCh is a non-aromatic amino acid (Ala), which is a common feature of TTX-resistant channels. These findings suggest that whether the amino acid at the 3^rd position in the SS2 region of repeat I is aromatic or non-aromatic determines the TTX sensitivity of Na channels, not the overall structure of the channel.