A concatenated cDNA was made that comprised four contiguous copies of the rat brain potassium channel subunit Kv1.1. Currents were measured in oocytes that had been injected with in vitro transcribed RNA. A Pro residue was introduced by site-directed mutagenesis into the S4 transmembrane region of one of the four domains, at a position corresponding to that in which a Pro is found in voltage-dependent sodium and calcium channels. This substitution (L305P in any one domain) led to currents having a shallow activation curve, and an additional fast component of deactivation from strongly positive potentials. cDNAs with L305P substitution in two domains formed functional channels only if the domains were non-adjacent; the properties of the currents were similar to the wild-type concatemer. In both cases, 100-1000 x more RNA was injected to obtain maximal currents similar to those seen with the wild-type concantemer. The point mutation Y379V is known to reduce the blocking potency of extracellular tetraethylammonium; this residue from each of the four domains is exposed at the outer mouth of the pore because the progressive introduction of 1, 2, 3 and 4 such mutations causes progressive reductions in tetraethylammonium sensitivity. The Y379V mutation was introduced into concatenated cDNAs with two non-adjacent Pro-containing domains; the sensitivity to TEA of the resulting currents showed that the Pro-containing subunits did not contribute to the pore-forming part of the channel. The results suggest that channels can form with a Pro substitution in a single S4 domain, but they require strong depolarization to open and deactivate rapidly upon repolarization. With Pro substitutions in two domains, channels appear to be formed as a multimerized concatemer, in which the Pro-containing domains are excluded from pore formation.