Human cytomegalovirus (HCMV) encodes four putative G protein-coupled receptors, including pUL78, whose rodent orthologues are known to be important for replication and spread in their hosts. To investigate the mechanism by which pUL78 contributes to viral replication and pathogenesis, we generated a derivative of the TB40/E clinical isolate of HCMV that is unable to express the receptor. Consistent with previous findings using laboratory strains of the virus, the mutant replicated normally in fibroblasts. Although laboratory strains are restricted to growth in fibroblasts, clinical isolates grow in many cell types, including epithelial and endothelial cells, in which the pUL78-deficient TB40/E derivative exhibited a growth defect. Infection with the mutant virus resulted in a significant decrease in viral RNA and protein expression. Although there was no difference in binding of the virus to the cell, we detected a delay in the entry and subsequent delivery of virion DNA and protein to the nuclei of epithelial cells following infection with the UL78 mutant virus. Taken together, our results demonstrate that pUL78 supports infection at a point after binding but before entry in epithelial cells, a cell type important for in vivo viral replication and spread.