The inward Na+ current underlying the action potential in nerve is terminated by inactivation. The preceding report shows that deletions within the intracellular linker between domains III and IV remove inactivation, but mutation of conserved basic and paired acidic amino acids has little effect. Here we show that substitution of glutamine for three clustered hydrophobic amino acids, Ile-1488, Phe-1489, and Met-1490, completely removes fast inactivation. Substitution of Met-1490 alone slows inactivation significantly, substitution of Ile-1488 alone both slows inactivation and makes it incomplete, and substitution of Phe-1489 alone removes inactivation nearly completely. These results demonstrate an essential role of Phe-1489 in Na(+)-channel inactivation. It is proposed that the hydrophobic cluster of Ile-1488, Phe-1489, and Met-1490 serves as a hydrophobic latch that stabilizes the inactivated state in a hinged-lid mechanism of Na(+)-channel inactivation.