To begin to study the molecular bases that determine the selective interaction of the beta-subunits of voltage-gated K+ channels with alpha-subunits observed in situ, we have expressed these polypeptides in transfected mammalian cells. Analysis of the specificity of alpha/bet a-subunit interaction indicates that both the Kvbeta1 and Kvbeta2 beta-subunits display robust and selective interaction with the five members of the Shaker-related (Kv1) alpha-subunit subfamily tested. The interaction of these beta-subunits with Kv1 alpha-subunits does not require the beta-subunit N-terminal domains. Thus, the previously observed failure of N-terminal mutants of Kv beta1 to modulate inactivation kinetics of Kv1 family members is not simply due to a lack of subunit interaction. Interaction of these beta-subunits with members of two other subfamilies (Shab- and Shaw-related) could not be detected. Somewhat surprisingly, a member of the Shal-related subfamily was found to interact with beta-subunits; however, this interaction had biochemical characteristics distinct from the beta-subunit interaction with Kv1 family members. In all cases, Kvbeta1 and Kvbeta2 exhibited indistinguishable alpha-subunit selectivity. These studies point to a selective interaction between K+ channel alpha- and beta-subunits mediated through conserved domains in the respective subunits.