|When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (Delta F) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Phys iol, 112:391-408.). We attached TMRM at the same sites [corresponding to M356C and X359C in die wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3-S4 linker. III the deletion mutant, the maximal Delta F/F seen was diminished 10-fold, and the Delta F at M356C because pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3-S4 linker. The residual Delta F showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV: During activating voltage steps from a holding potential of -90 mV, the fluorescence lagged considerably behind tl-Ie movement of gating char ge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a Delta F at extreme hyperpolarizations (more negative than -90 mV) only A signal from the inter action with this group in the wt S3-S4 linker channel (at L361C) correlated with gating char-ge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic Delta F as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.