Mouse cytomegalovirus (MCMV) functions expressed at the beginning of the early phase of the viral replication cycle interfere with the major histocompatibility complex (MHC) class I-restricted pathway of antigen presentation (M. J. Reddehase, M. R. Fibi, G. M. Keil, and U. H. Koszinowski, J. Virol. 60:1125-1129, 1986; M. Del Val, K. Münch, M. J. Reddehase, and U. H. Koszinowski, Cell 58:305-315, 1989). Nascent MHC class I heavy chains associate with beta 2-microglobulin and peptide, but the assembled trimolecular complex is retained in the endoplasmatic reticulum/cis-Golgi compartment (M. Del Val, H. Hengel, H. Häcker, U. Hartlaub, T. Ruppert, P. Lucin, and U. H. Koszinowski, J. Exp. Med. 176:729-738, 1992). To locate the responsible genomic region, the cytoplasmic retention of MHC class I molecules after injection of MCMV DNA was tested. The function was mapped to the HindIII E fragment. A recombinant MCMV deletion mutant delta MS94.5 lacking 15.8 kb in HindIII-E was constructed. Restoration of MHC class I molecule maturation and recognition of antigenic peptides by cytolytic T lymphocytes during the first hours of the early phase in mutant virus-infected cells proved the correct location to a 6.8-kb region in the HindIII E fragment. At later stages of the early phase, membrane-resident MHC class I molecules and cytolytic T lymphocyte recognition disappeared in delta MS94.5 mutant virus-infected cells. These results demonstrate that more than one early-gene function of MCMV affects the MHC class I pathway of antigen presentation. The redundant MHC class I-reactive functions target the transport of MHC class I molecules at different steps.