Ribosylation of 2-chloro-5(6)-nitrobenzimidazole (3) gave 2-chloro-5-nitro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)benzimidazol e (4a) and 2-chloro-6-nitro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)benzimidazol e (4b) as a mixture of positional isomers. Subsequent hydrogenation of this mixture over Raney Nickel afforded the corresponding 5-amino and 6-amino derivatives 5 and 6. At this stage the products were readily resolved via silica column chromatography into pure isomeric forms, and the pure isomers 5 and 6 were diazotized with tert-butyl nitrite and cupric chloride to furnish the isomerically pure 5-chloro derivative 2a and 6-chloro derivative 2b. Deprotection of 5, 6, 2a, and 2b with methanolic ammonia yielded the free nucleosides 5-amino-2-chloro-1-(beta-D-ribofuranosyl)benzimidazole (7), 6-amino-2-chloro-1-(beta-D-ribofuranosyl)-benzimidazole (8), 2,5-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (9), and 2,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (10), respectively. Treatment of 10 with thiourea afforded 6-chloro-1-(beta-D-ribofuranosyl)benzimidazole-2-thione (14). Alkylation of 14 with methyl iodide and benzyl bromide gave good yields of the corresponding 2-methylthio (12) and the 2-benzylthio (13) analogs. The synthesis of 6-chloro-2-methoxy-1-(beta-D-ribofuranosyl)benzimidazole (11) was accomplished by the treatment of 2b with sodium methoxide in methanol. A difference NOE spectroscopic experiment was conducted to allow unequivocal assignment of regiochemistry to the positional isomers 5 and 6. Evaluation of compounds for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 revealed that the heterocycle 3 was active against both viruses but also was cytotoxic. Only the dichloro compounds 9 and 10 were weakly active against HCMV and noncytotoxic in their antiviral dose range. These data further substantiate the conclusion that activity against HCMV at noncytotoxic concentrations by benzimidazole ribonucleosides requires a halogen not only at the 2-position, but also more than one halogen on the benzene moiety.