The importance of asparagine-linked glycosylation in the cell surface expression of several class I molecules was examined. C57BL/6 (B6) T cell blasts were treated with tunicamycin (TM), an antibiotic that inhibits N-linked glycosylation. The levels of various class I molecules on these cells were examined by flow cytometry and were compared to the levels of the same molecules on untreated cells. A 12-hr TM treatment did not significantly alter the levels of H-2Kb, Db, or Qa-2; however, such treatment decreased the surface expression of the Qa-1b allelic product to undetectable levels. A time-course study indicated that a decrease in the level of Qa-1.2 expression was apparent after only 4 hr of TM treatment. An examination of T cell blasts prepared from mouse strains possessing the Qa-1a, Qa-1(c), and Qa-1(d) alleles indicated that all allelic products of this locus demonstrated a marked decrease in cell surface expression on TM treatment, whereas other class I molecules (H-2K(s), TL) exhibited slight or no decrease. Two-dimensional polyacrylamide gel electrophoresis analysis of immunoprecipitates from detergent lysates of surface-iodinated TM-treated B6 blasts revealed the presence of the unglycosylated form of the H-2Kb molecule of the cell surface. No such form of the Qa-1.2 molecule could be detected by similar analysis. To establish that the above observations were not simply a result of the inability of the Qa-1-specific alloantisera to react with the unglycosylated Qa-1 molecule, lysates of surface-iodinated B6 blasts were digested with endoglycosidase F, which cleaves N-linked carbohydrate moieties. Immunoprecipitation analysis indicated that the antisera could react with the unglycosylated form of the Qa-1 molecule. These results indicate that N-linked glycosylation has differential importance in the cell surface expression of class I molecules.