The endoplasmic reticulum resident protein calnexin interacts with several glycoproteins including class I MHC molecules. Calnexin is thought to retain free class I heavy chains and/or promote their folding and assembly with beta 2-microglobulin and peptide ligand. Whereas with other glycoproteins, Asn-linked glycans seem to be involved in calnexin association, with class I molecules the transmembrane region has been implicated. To critically define the structures on class I molecules that determine their interaction with calnexin, we have studied carbohydrate-deficient and transmembrane-variant class I molecules. Carbohydrate-deficient class I molecules were found to accumulate intracellularly in an open, non-beta 2-microglobulin-associated conformation. However, open as well as conformed class I molecules showed significant calnexin association whether they were aglycosylated or fully glycosylated. Thus, carbohydrate moieties may be necessary for efficient class I folding, but are not required for calnexin association. Calnexin was also found associated with a soluble class I molecule that has a truncated transmembrane segment, demonstrating that membrane attachment of class I is not required for interaction with calnexin. Finally, two isoforms of the class Ib molecule Q7b were compared. Unexpectedly, the glycosylphosphatidylinositol-anchored Q7b isoform was found associated with calnexin, whereas the soluble Q7b isoform was not calnexin associated. These comparisons of Q7b isoforms implicate the class I-connecting peptide segment and not the transmembrane region as a site of interaction with calnexin.