Recently several cell lines have been identified with mutations in a major histocompatibility complex (MHC)-linked protein that lead to defects in class II-restricted antigen presentation and a defect in the formation of class II SDS-stable dimers. The defect in these cells has recently been shown to result from the inability to express the MHC-encoded nonclassical class II molecule called DM. To further examine the role of DM in class II-restricted antigen presentation, we asked if this defect would equally affect different allelic and species variants of class II molecules. To investigate this, we transfected the parent cell lines T1 and 8.1.6 and their respective antigen presentation mutants T2 and 9.5.3 with the genes encoding I-Ad and examined the derived transfectants for their ability to present antigen, the conformation of I-Ad at the cell surface, association of I-Ad with invariant chain (Ii), and the ability to form I-Ad SDS-stable dimers. The lack of functional DM expression did not affect any of the anti-I-Ad monoclonal antibody (mAb) epitopes tested or the ability of I-Ad to associate and dissociate with Ii. Surprisingly, these studies also revealed that the antigen presentation defect observed for DR in the 9.5.3 cells did not compromise I-Ad-restricted antigen presentation. In addition, we found that the level of SDS-stable dimer formation did not correlate with antigen presentation capacity for I-Ad and that the amount of SDS-stable I-Ad dimer depends on the cellular context in which the class II molecule is expressed. Our results suggest that the ability to form SDS-stable dimer is not strictly correlated with class II-restricted antigen presentation. Finally, when two allelic forms of murine class II molecules were compared in the defective T2 cell line, it was found that I-Ak but not I-Ad forms SDS-stable dimers equivalent to that seen in the parental cell lines. Overall, our results suggest that DM may modulate rather than play a requisite role in I-Ad-restricted antigen presentation and SDS-stable dimer formation and that dependency on DM may be allele or species specific.