The lateral mobility of cell membrane glycoproteins is often restricted by dynamic barriers. These barriers have been detected by measurements of fluorescence photobleaching and recovery (FPR) and barrier-free path (BFP). To define the location and properties of the barriers, we compared the lateral mobility, measured by FPR and BFP, of wild-type class I major histocompatibility complex (MHC) membrane glycoproteins with the lateral mobility of mutant class I MHC glycoproteins truncated in their cytoplasmic domains. Mutants with 0 or 4 residues in the cytoplasmic domain were as mobile as lipid-anchored class I MHC molecules, molecules whose lateral mobility is relatively unrestricted by barriers. In contrast, mobility of class I MHC molecules with 7-residue cytoplasmic domains was as restricted as mobility of class I molecules with full-length, 31-residue cytoplasmic domains. Though some of the difference between the mobilities of mutants with 4- or 0-residue domains and the other class I molecules may be due to differences in the net charge of the cytoplasmic domain, FPR measurements of the mobility of molecules with 7-residue domains show that length of the cytoplasmic domain has an important influence on the lateral mobility. Model calculations suggest that the barriers to lateral mobility are 2-3 nm below the membrane bilayer.