Heag1 and heag2 channels are members of the eag family of human potassium channels. These channels have high sequence similarity, but differ in their electrophysiological properties, such as steady state activation (Ju & Wray, 2002). In this study, we have investigated the molecular regions that contribute to the differences in steady state activation between heag1 and heag2 channels, focussing here on which parts of the membrane-spanning regions S1 to S6 are involved. One might expect that differences in activation are due to the voltage sensor region S1-S4. For this, chimeras between heag1 and heag2 were constructed by restriction digest and subsequent ligation, transferring corresponding regions between the channels. The chimeras were expressed in Xenopus oocytes and currents were recorded two days later at room temperature. Oocytes were depolarized from a holding potential of -80 mV to test potentials from -70 mV to +70 mV. Conductance-voltage (G-V) curves were obtained and fitted to the Boltzmann equation. Controls with wild type channels were used in the same batch of oocytes as the chimeras. Chimera I had the S1 to S6 transmembrane region of heag2 (residues 138-549) replaced by heag1. For this chimera, the Boltzmann slope parameter k was similar to that for wild type heag1 channel, but significantly different from that for wild type heag2 channel (chimera I: 13.7 ± 1.0 mV, n=8; wild type heag2: 42.0 ± 3.1 mV, n=4, P<0.05, Student's unpaired t-test). This confirms that indeed as expected, the S1-S6 region is involved in determining steady state activation in heag channels. To determine which parts of the S1-S6 region are involved, we studied further constructs. Chimera II had the S1 to S4 transmembrane region of heag2 (residues 138-331) replaced by heag1. The slope parameter k for this chimera was intermediate in magnitude between wild types heag1 and heag2; for the chimera, k was 32.9 ± 1.5 mV, n=8, a value significantly different (P<0.05) from both wild type heag1 (23.3 ± 1.4 mV, n=7) and heag2 (50.0 ± 3.8 mV, n=8). Chimera III, with the S5 to S6 region of heag2 (residues 332-549) replaced by heag1 showed a k value (13.5 ± 0.8 mV, n=8) that was closest to, but not identical to, that for heag1 (23.2 ± 1.0 mV, n=7) rather than heag2 (40.4 ± 2.5 mV, n=7). Taken together, the data show that, residues within the whole of the S1-S6 transmembrane region are involved in determining differences in steady state activation between heag1 and heag2 channels; both S1-S4 and S5-S6 regions are involved in the effect. Thus it is not only the voltage sensor, but also the pore region that determines voltage sensitivity of activation.