In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.