Voltage-gated Na+ channels are essential for the normal electrical excitability of neuronal and striated muscle membranes. Distinct isoforms of the Na+ channel alpha-subunit have been identified by molecular cloning, and their functional attributes have been defined by heterologous expression coupled with electrophysiological recording. Two closely related Na+ channel alpha-subunit isoforms, hH1 (human heart) and hSkM1 (human skeletal muscle), exhibit differences in their inactivation properties and in their response to the coexpressed beta 1-subunit. To localize regions that contribute to inactivation and to beta 1-subunit response, we have exploited these functional differences by studying chimeric channels composed of segments from both hH1 and hSkM1. Chimeras in which one or more of the cytoplasmic interdomain regions (ID1-2, ID2-3, and ID3-4) were exchanged between hH1 and hSkM1 exhibit inactivation properties identical with the background channel isoform, suggesting that these regions are not sufficient to cause gating differences. In contrast, inactivation properties of chimeras composed of approximately equal halves of the two channel isoforms were intermediate between hH1 and hSkM1. Furthermore, the response to the coexpressed beta 1-subunit was dependent on structures located in the carboxy-terminal half of the alpha-subunit, although domains D3, D4, and the carboxy terminal are not singularly responsible for this effect. These data indicate that inactivation differences between hH1 and hSkM1 are determined by multiple alpha-subunit domains.