Protein targeting mechanisms in flagellated protozoan parasites have received considerable interest because of a huge bias in these organisms toward the glycosylphosphatidylinositol anchor as a mechanism for the membrane attachment of cell surface macromolecules. In this study, the trafficking of invariant surface glycoprotein 65 (ISG65), a family of type I transmembrane proteins, was examined. Analysis of the C-terminal domains of ISG65 family members demonstrated a high level of conservation and, in particular, the presence of three lysine residues contained within the cytoplasmic tails of all ISG65s. ISG65 was expressed on the cell surface, in agreement with earlier work, but an intracellular pool of ISG65 was also detected within a Rab5A early endosome. Transplantation of the C-terminal 74 amino acids of ISG65 (encompassing the 23 C-terminal residues of the extracellular domain, the transmembrane peptide, and the cytoplasmic domain) onto the N-terminal domain of BiP (BiPN) was sufficient to target the chimera to the same internal compartments as native ISG65. Further, site-directed mutagenesis indicated that the cytoplasmic tail was required for endoplasmic reticulum exit and that at least two of the cytoplasmic domain lysine residues are needed for endosomal targeting, as removal of all three led to surface expression. Kinetic measurements demonstrate that the BiPN fusion protein (containing the ISG65 C terminus) has a short half-life, indicating rapid turnover. In contrast, BiPN fusion proteins containing a glycosylphosphatidylinositol anchor instead of the ISG65 C-terminal region are stably expressed on the surface, confirming the requirement for the ISG65 sequence for endosomal targeting. We suggest that the lack of surface expression of the BiPN-ISG65 fusion protein is likely due to more efficient internalization compared with ISG65. Taken together, these data demonstrate the presence of a lysine-dependent endocytosis signal in the ISG65 family.