Loops connecting core secondary structure elements of proteins are highly variable in length and sequence between homologs; as such they are often thought to have little or no participation in the function of many proteins. In the case of voltage-gated potassium channels, truncation of the loop connecting the S3 and S4 helices has been demonstrated to alter voltage sensitivity (Gonzalez et al. (2000) J. Gen. Physiol. 115: 193-208 and (2001) P.N.A.S. 98: 9617-23). We have studied the effect of replacing this loop with homopolymers of different length and composition, and compared these effects with results of molecular dynamics simulations of these short peptide sequences. After each simulation, a free energy profile was plotted as a function of end-to-end distance of the isolated loops, thus allowing a correlation between loop dynamics and voltage-sensitive opening and closing. Constraints on the distance between the C-terminus of the S3 helix and the N-terminus of the S4 helix affect both equilibrium and kinetic properties of mouse Kv1.2. The shortest loops, consisting of two amino acids, all lead to a strong positive shift in the V50 values for the channels, whereas long loops, where the end-to-end distance can exceed 1.5 nm, have smaller effects on the V50 while having significant effects on the kinetics of channel opening. Glutamate linkers cause significant kinetic slowing, but proline loops four residues in length show the most positive V50 and longest 10-90% rise time. Channels constructed with short loops of proline and serine also appear to conduct small amounts of inward current at a wide range of voltages prior to outward conductance. The G/V-derived Boltzmann slope factor was not significantly affected by altering the S3-S4 loop sequence.