We constructed chimeras between the rapidly activating Kv1.2 channel and the slowly activating Kv2.1 channel in order to study to what extent sequence differences within the S1-S4 region contribute to the difference in activation kinetics. The channels were expressed in Xenopus oocytes and the currents were measured with a two-microelectrode voltage-clamp technique. Substitution of the S1-S4 region of Kv2.1 subunits by the ones of Kv1.2 resulted in chimeric channels which activated more rapidly than Kv2.1. Furthermore, activation kinetics were nearly voltage-independent in contrast to the pronounced voltage-dependent activation kinetics of both parent channels. Systematic screening of the S1-S4 region by the replacement of smaller protein parts resolved that the main functional changes generated by the S1-S4 substitution were generated by the S2 and the S3 segment. However, the effects of these segments were different: The S3 substitution reduced the effective gating charge and accelerated both a voltage-dependent and a voltage-independent component of the activation time course. In contrast, the S2 substitution accelerated predominantly the voltage-dependent component of the activation time course thereby leaving the effective gating charge unchanged. It is concluded that the S2 and the S3 segment determine the activation kinetics in a specific manner.