Class I MHC Ag presentation and cell surface expression largely depend on peptide transport into the ER/cis-Golgi by TAP, the transporter associated with Ag processing. Despite this dependency, class I MHC molecules are expressed at low levels on the surface of TAP-deficient T2 cells. We studied the peptide dependency of HLA-B7 expression in transfected T2 cells. HLA-B7 expression was affected by mutations at 19 out of 23 peptide-binding groove residues, but not by nine mutations outside of the peptide-binding groove. T2 cell surface HLA-A2, -B7, and -B51 had similar stabilities, and approximately half of these class I molecules had a long t1/2 consistent with tight peptide binding. Using metabolically labeled T2 cells, HLA-A2-bound peptide eluted as five prominent peaks, but HLA-B7-bound peptide was not detected. In contrast, HLA-B7-eluted peptides were detected spectrophotometrically. These data suggest that HLA-A2 and HLA-B7 molecules utilize distinct TAP-independent peptide supply mechanisms to different degrees. Equivalent amounts of HLA-B7 from TAP- and TAP+ cells yielded similar amounts of peptide, which had the characteristic HLA-B7 peptide-binding motif. The dependency of HLA-B7 cell surface expression on peptide-binding groove residues, the stability of cell surface class I molecules, and the ability to detect HLA-B7-bound peptide indicate that the low level expression on T2 cells is largely peptide dependent. TAP-independent peptide Ag presentation may allow immune recognition of intracellular pathogens that interfere with TAP-dependent peptide transport.