The immediate early protein ICP47 of the Herpes simplex virus is known to block the human transporter associated with antigen processing (TAP), thereby creating a TAP-deficient phenotype in any human cell transfected with the corresponding cDNA. Exploiting this inhibitory activity, we constructed a selection of human cell lines each co-expressing one of the cDNAs of human leukocyte antigen (HLA) class I alleles HLA-A*1101, A24, A*3101, A*6601, B8 and B*1516, and the cDNA encoding the ICP47 molecule. The cell lines generated showed diminished HLA class I surface expression and the inhibition of the TAP function was confirmed in peptide translocation assays. The addition of specific exogenous peptide ligands restored the expression of the corresponding HLA class I molecules. Thus, the ICP47 transfectants provide us with a tool to closely examine peptide-HLA class I interactions, to confirm HLA class I ligand motifs and to test peptides predicted to bind.