In a study of the infection of mouse embryo fibroblasts with murine cytomegalovirus (MCMV), we found that plaque number is directly related to virus concentration and not to the total amount of virus contained in the inoculum. These results suggested that virus binding was reversible and that during infection a binding equilibrium is established which limits the amount of bound virus. Further analysis revealed three categories of plaque based on reversibility after virus adsorption. One group was removed simply by washing cell monolayers after virus removal. A second group of plaques was lost gradually with time, giving complete reversal after 5 min at 37 degrees C. The rate of reversal was temperature dependent, and probably represented true virus dissociation. The final group was irreversible plaques, the number of which increased with increasing infection time. The number of reversible plaques remained constant with time of infection, and represented about 70% of the total plaques after 1 h of virus adsorption. Centrifugation of the virus inoculum onto the fibroblast monolayer at 1000 X g increased plaque numbers up to 100-fold, but had little effect on plaque number when carried out after the virus inoculum was removed. In contrast centrifugation increased the number of reversible plaques, suggesting an increase in the number of virus particles attached to the cell monolayers. We suggest that centrifugation enhances MCMV infection by three mechanisms related to reversibility of binding: (1), it increases the rate of virus association; (2), it decreases the rate of dissociation; (3), by increasing the length of time each virus particle is bound it increases the probability of virus being taken into the cell.