We have already shown that in species with highly polymorphic major histocompatibility complex (MHC) class I molecules (human, mouse) no functional polymorphism of the peptide transporters TAP1 and TAP2 is detectable (Lobigs and Müllbacher 1993). Investigating the antigen-presentation machinery of the class I MHC monomorphic Syrian hamster using mouse MHC class I expression via recombinant vaccinia viruses (VV) we found that six hamster cell lines fall into two phenotypic classes. four cell lines (HaK, FF, MF-2, and HT-1) showed no defect in expressing four different H2 class I molecules (Kk, Kd, Kb, Dd) and the appropriate VV peptide recognized by mouse VV-immune cytotoxic T (Tc) cells on the cell surface. Two cell lines (BHK-21 and NIL-2) expressed Dd and Kb in association with VV peptides as recognized by VV-immune, H2-restricted Tc cells but not Kk and Kd. However, Kd was expressed on the cell surface, as shown by fluorescence-activated cell sorter (FACS) analysis and alloreactive Tc-cell recognition. Kk is only surface-expressed in these two cell lines when superinfected with two VV recombinants encoding rat TAP1 (VV-mtp1) and TAP2 (VV-mtp2). Superinfection with VV-mtp1 and VV-mtp2 rendered both cell lines, after infection with either VV-Kk and VV-Kd, susceptible to lysis by either Kk- or Kd-restricted VV-immune Tc cells. Thus Syrian hamster cell lines express functionally polymorphic peptide transporters. The TAP2 gene from FF cells was cloned and sequenced; comparison with human, mouse, and rat TAP2 sequences show 78%, 88% and 87% similarity, respectively.