The oligomeric complex formed by major histocompatibility complex (MHC) class II alpha and beta chains and invariant chain (Ii) assembles in the endoplasmic reticulum and is then transported via the Golgi complex to compartments of the endocytic pathway. When Ii alone is expressed in CV1 cells it is sorted to endosomes. The Ii cytoplasmic tail has been found to be essential for targeting to these compartments. In order to characterize further the signals responsible for endosomal targeting, we have deleted various segments of the cytoplasmic tail. The Ii mutants were transiently expressed and the cellular location of the proteins was analyzed biochemically and morphologically. The cytoplasmic tail of Ii was found to contain two endosomal targeting sequences within its cytoplasmic tail; one targeting sequence was present within amino acid residues 12-29 and deletion of this segment revealed the presence of a second endosomal targeting sequence, located within the first 11 amino acid residues. The presence of a leucine-isoleucine pair at positions 7 and 8 within this sequence was found to be essential for endosomal targeting. In addition, the presence of this L-I motif lead to accumulation of Ii molecules in large endosomal vacuoles containing lysosomal marker proteins. Both wild type Ii and Ii mutant molecules containing only one endosomal targeting sequence were rapidly internalized from the plasma membrane. When the Ii cytoplasmic tail was fused to the membrane-spanning region of neuraminidase, a resident plasma membrane protein, the resulting chimera (INA) was found in endocytic compartments containing lysosomal marker proteins. Thus the cytoplasmic tail of Ii is sufficient for targeting to the endocytic/lysosomal pathway.