Invariant chain (Ii) has been shown to play a significant part in the assembly of MHC class II molecules. Ii also binds to MHC class I, although it is not known when this first occurs or whether it can affect class I assembly. Our examination of lysates of L(d)-transfected T2 cells showed that Ii bound intracellularly to folded, but not to open, forms of MHC class I. Furthermore, addition of peptides to the lysates dissociated Ii from the Ii-folded MHC class I complex. Thus, unlike other known chaperones, Ii associates only with folded, peptide-free class I molecules. To determine whether Ii can affect MHC class I transport and surface expression, we used both wild-type Ii and a mutant Ii that lacked the endosomal targeting sequence. Neither Ii nor Ii(Delta 20) increased the rate of MHC class I migration; however, Ii and (to a greater extent) Ii(Delta 20) increased cell surface expression of MHC class I. In HeLa cells, this effect was allele-specific, affecting HLA-A28 more than -B75. Ii also increased the surface expression of K(b) more than D(b) on Panc02 pancreatic adenocarcinoma cells. Neither form of Ii was detectable at the cell surface with MHC class I, indicating that Ii had exercised its effect on class I intracellularly. In total, these data suggest that Ii can bind peptide-free folded class I/beta(2)m heterodimers, but not open MHC class I heavy chains, in the endoplasmic reticulum, and that Ii can facilitate the surface expression of the MHC class I molecule.