We have studied the role of the Zn²⁺ ion-coordinating C3H1 motif of the N-terminal tetramerization domain (T1) domain in homomeric and heteromeric assembly of the voltage-activated (Kv) potassium channels. The T1 domain has been shown previously to play a role in the homomeric assembly of Kv2, Kv3 and Kv4 channels, but the mechanisms controlling specific heteromeric assembly are still elusive. Using a yeast-two-hybrid assay, we found that substitution of histidine-105 in the C3H1 motif of Kv2.1 by valine (H105V) or arginine (H105R) disrupted the interaction of the T1 domain of Kv2.1 with the T1 domains of both Kv6.3 and Kv6.4, whereas the interaction of the mutated T1 domain of Kv2.1 with itself was unaffected. Using fluorescence resonance energy transfer (FRET), interaction could be detected between the subunits Kv2.1/Kv2.1, Kv2.1/Kv6.3 and Kv2.1/Kv6.4. Reduced FRET signals were obtained after co-expression of Kv2.1^H105V or Kv2.1^H105R with Kv6.3 or Kv6.4. Wild-type Kv2.1 but not Kv2.1^H105V could be co-immuno-precipitated with Kv6.4. Co-expression of wild-type Kv2.1 with Kv6.3 or Kv6.4 in CHO-K1 cells had pronounced effects on the time course of activation, deactivation and inactivation and on the voltage-dependence of activation of Kv2.1, whereas co-expression of the mutants Kv2.1^H105R or Kv2.1^H105V with Kv6.3 or Kv6.4 did not modify the biophysical properties of the mutant channels at all. These findings were confirmed by co-expression of the same mutants in Xenopus oocytes. Co-expression experiments with dominant-negative pore mutants (GYG -> AYA) in Xenopus oocytes showed that Kv2.1^H105R subunits do not co-assemble with Kv6.3. Our results strongly suggest that histidine-105 in the T1 domain of Kv2.1 is required for functional heteromerization with members of the Kv6 subfamily. We conclude from our findings that Kv2.1 and Kv6.x subunits have complementary T1 domains which control selective heteromerization.