We have previously reported that 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(beta-D-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5'-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [(3)H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5'-monophosphate (BDCRB-5'-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5'-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5'-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5'-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5'-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5'-deoxy analog (5'-dTCRB) did not affect viral DNA synthesis, but 5'-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5'-dTCRB and the 5'-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5'-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5' position for antiviral activity.