Antibodies that neutralize cytomegalovirus (CMV) entry into fibroblasts are predominantly directed against epitopes within virion glycoproteins that are required for attachment and entry. However, the mechanism of CMV entry into epithelial and endothelial cells differs from fibroblast entry. Using assays that simultaneously measured neutralizing activities against CMV entry into fibroblasts and epithelial cells, we found that human immune sera and CMV-hyperimmuneglobulins have on on average 48-fold higher neutralizing activities against epithelial cell entry compared to fibroblast entry, suggesting that natural CMV infections elicit neutralizing antibodies that are epithelial entry-specific. This activity could not be adsorbed with recombinant gB. The Towne vaccine and the gB/MF59 subunit vaccine induced epithelial entry-specific neutralizing activities that were on on average 28-fold (Towne) or 15-fold (gB/MF59) lower than those observed following natural infection. These results suggest that CMV vaccine efficacy may be enhanced by the induction of epithelial entry-specific neutralizing antibodies.