Antiviral activities of acyl derivatives (3′-
O
-octanoyl and 3′-
O
-decanoyl) of 2,2′-anhydro-1-β-
d
-arabinofuranosylcytosine (cyclo-C) and 1-β-
d
-arabinofuranosylcytosine (Ara-C) were compared with other antiviral nucleosides, and some biological characteristics of the antiviral activity were investigated. Among those synthesized acyl derivatives, 3′-
O
-decanoyl ara-C was the most active against deoxyribonucleic acid viruses, with an activity comparable to that of Ara-C. Acyl derivatives of cyclo-C were somewhat less active than their Ara-C counterparts. In the value of therapeutic index, 1-β-
d
-arabinofuranosyladenine was superior to the others, followed by 5-iodo-2′-deoxyuridine. In comparing the sensitivity of two serotypes of herpes simplex virus it was found that Ara-C and its ester, as well as its cyclo-C counterpart, were more active against the type 2 than the type 1 strain. The activity of 3′-
O
-decanoyl Ara-C, like that of its parent, was diminished by treatment with cytidine deaminase from mouse kidney, but 3′-
O
-decanoyl cyclo-C was resistant to this treatment. In comparative studies of 3′- and 5′-
O
-acyl Ara-C's, antivaccinia virus activity of 3′-
O
-palmitoyl Ara-C was significantly superior to its 5′-counterpart. The inhibitory activity of 5′-
O
-decanoyl Ara-C was markedly reduced by the presence of a threefold molar excess of eserine sulfate, a choline esterase inhibitor, whereas the 3′-acyl Ara-C was not affected by the inhibitor in any combination. This result indicates that enzymatic hydrolysis of the 3′-ester to Ara-C, which is inhibited by eserine sulfate, did not occur in this cell culture.