Invariant chain (Ii) association with MHC class II molecules is strongly dependent upon interaction of CLIP (Ii exon 3, residues 81 - 104) with the peptide binding groove of the class II dimer. This dominant interaction does not adequately explain, however, the efficient association of Ii with class II molecules of diverse allelic and isotypic origin, which have markedly different affinities for synthetic peptides corresponding to CLIP. In agreement with other recent observations, we demonstrate here that class II molecules with occupied binding sites unable to engage CLIP maintain association with Ii in mild detergent. The association is direct and not mediated through unoccupied class II chains bound to properly assembled and loaded class II dimers, nor is it mediated through chaperones. The site of this CLIP-independent binding has been mapped using truncation mutants and an Ii-human transferrin receptor chimeric protein to the transmembrane segment of Ii. The existence of multiple low-affinity sites of interaction between MHC class II and Ii helps explain how effective occupancy of all newly synthesized class II molecules can occur despite substantial variations in the strength of CLIP-dependent association that arise from class II binding domain polymorphism. These data establishing a site of Ii-MHC class II association N-terminal to CLIP also provide new insight into the possible functional relationship between the sequential endocytic proteolysis of Ii from its C terminus and a series of contact sites with MHC class II molecules spread from the transmembrane region through to the tip of the lumenal segment of Ii.