The human cytomegalovirus (HCMV) U(L)26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the U(L)26 protein that contribute to viral infection, we created a panel of site-directed U(L)26 mutant viruses and assessed their impact on phenotypes attributed to U(L)26. We find that the C-terminal 38 amino acids of the U(L)26 protein are absolutely necessary for U(L)26 function. A stop-insertion mutant that produced a truncated U(L)26 protein lacking this region behaved identically to U(L)26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of U(L)26 packaged into the virion resulting in reduced delivery of U(L)26 to newly infected cells. Further, this C-terminal truncated U(L)26 exhibited substantially reduced nuclear localization compared to wildtype U(L)26. Translation of U(L)26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of U(L)26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of U(L)26, are also important for the function of the U(L)26 protein. A viral mutant that produces only the short isoform of U(L)26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short U(L)26 isoform in the presence of the long U(L)26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the U(L)26 protein that contribute to HCMV infection.