The invariant chain (Ii) targets newly synthesized major histocompatibility complex class II complexes to a lysosome-like compartment. Previously, we demonstrated that both the cytoplasmic tail (CT) and transmembrane (TM) domains of Ii were sufficient for this targeting and that the CT contains two di-leucine signals, 3DQRDLI8 and 12EQLPML17 (Odorizzi, C. G., Trow-bridge, I. S., Xue, L., Hopkins, C. R., Davis, C. D., and Collawn, J. F. (1994) J. Cell Biol. 126, 317-330). In the present study, we examined the relationship between signals required for endocytosis and those required for lysosomal targeting by analyzing Ii-transferrin receptor chimeras in quantitative transport assays. Analysis of the Ii CT signals indicates that although 3DQRDLI8 is necessary and sufficient for endocytosis, either di-leucine signal is sufficient for lysosomal targeting. Deletions between the two signals reduced endocytosis without affecting lysosomal targeting. Transplantation of the DQRDLI sequence in place of the EQLPML signal produced a chimera that trafficked normally, suggesting that this di-leucine sequence coded for an independent structural motif. Structure-function analysis of the Ii TM region showed that when Ii TM residues 11-19 and 20-29 Were individually substituted for the corresponding regions in the wild-type transferrin receptor, lysosomal targeting was dramatically enhanced, whereas endocytosis remained unchanged. Our results therefore demonstrate that the structural requirements for Ii endocytosis and lysosomal targeting are different.