The predominant K+ channel in human T lymphocytes is Kv1.3, which inactivates by a C-type mechanism. To study assembly of these tetrameric channels in Jurkat, a human T-lymphocyte cell line, we have characterized the formation of heterotetrameric channels between endogenous wild-type (WT) Kv1.3 subunits and heterologously expressed mutant (A413V) Kv1.3 subunits. We use a kinetic analysis of C-type inactivation of currents produced by homotetrameric channels and heterotetrameric channels to determine the distribution of channels with different subunit stoichiometries. The distributions are well-described by either a binomial distribution or a binomial distribution plus a fraction of WT homotetramers, indicating that subunit assembly is a random process and that tetramers expressed in the plasma membrane do not dissociate and reassemble. Additionally, endogenous Kv1.3 current is suppressed by a heterologously expressed truncated Kv1.3 that contains the amino terminus and the first two transmembrane segments. The time course for suppression, which is maximal at 48 h after transfection, overlaps with the time interval for heterotetramer formation between heterologously expressed A413V and endogenous WT channels. Our findings suggest that diversity of K+ channel subtypes in a cell is regulated not by spatial segregation of monomeric pools, but rather by the degree of temporal overlap and the kinetics of subunit expression.