The role of beta-2-microglobulin (beta-2m) in murine cytomegalovirus (MCMV) infection of susceptible (H-2d) and resistant (H-2k) murine embryo fibroblasts (MEF) and peritoneal macrophages was evaluated using serum-free virus (SF-MCMV). The infectivity of SF-MCMV was significantly lower than virus propagated in serum, although the concentrations of virions were similar. Infection of cells with SF-MCMV was assessed by measuring the proportion of cells expressing viral antigens, the sizes of plaques formed in fibroblast monolayers and TCID50 titers. Infection of susceptible fibroblasts was significantly increased 1.6-4.7 fold by the addition of whole FCS, a <20 kDa FCS fraction, or purified human beta-2m. These supplements also significantly enhanced infection of susceptible macrophages and increased TCID50 titers by 3.5-10 fold in susceptible MEF. In relatively resistant H-2k cells, the TCID50 titer and the proportion of cells expressing viral antigens after infection with SF-MCMV were not affected by beta-2m or FCS, but plaque sizes were increased 2.5-3 fold in resistant BALB.K MEF.When human or murine beta-2m was added to infected cultures, immunogold electron microscopy revealed these proteins to be always associated extracellularly with the tegument material of disrupted multicapsid virions, but rarely with the envelope of intact virions. However, no murine beta-2m was found in association with the envelope or tegument of SF-MCMV. These relatively modest effects of beta-2m which were restricted to genetically susceptible cells, may be due to tegument-bound beta-2m facilitating infection by capsids, or the stabilisation of the conformation of Class 1 molecules by exogenous beta-2m, promoting MCMV binding to the target cell.