Using a combination of protease protection, glycosylation, and carbonate extraction assays, we have characterized the topogenic determinants encoded by Kv1.3 segments that mediate translocation events during endoplasmic reticulum (ER) biogenesis. Transmembrane segments S1, S2, S3, S5, and S6 initiate translocation, only S1 and S2 strongly (>60%) anchor themselves in the membrane, S5 exhibits signal anchor activity and contains a cryptic cleavage site, and S3 and S6 fail to integrate into the membrane. Elongation of each single-transmembrane construct to include multiple transmembrane segments alters integration and translocation efficiencies, indicating that multiple topogenic determinants cooperate during Kv1. 3 topogenesis and assembly. Several surprising findings emerged from these studies. First, in the presence of T1, the N-terminal recognition domain, S1 was unable to initiate either translocation or membrane integration. As a result, S2 likely functions as the initial signal sequence to establish Kv1.3 N-terminus topology. Second, S4 independently integrates into the membrane. Third, S6 plus the C-terminus of Kv1.3 is a secretory protein but can be converted to a membrane-integrated protein with a correctly oriented, cytosolic C-terminus by linking S6 to S5 and the pore loop. These results have implications for the role of the N-terminus in Kv biogenesis and on the mechanisms of dominant negative suppression of Kv1.3 by truncated Kv1.3 fragments [Tu et al. (1996) J. Biol. Chem. 271, 18904-18911].