2,2'-Anhydro-1-(3'-O-acetyl-beta-D-arabinofuranosyl)-5-iodocytosine (anhydro-araIC 3'-acetate), previously synthesized and isolated as a crude product by Moffatt and his coworkers, was purified and characterized. The availability of pure anhydro-araIC 3'-acetate made possible a comparative study of the antineoplastic, antiviral and biochemical potencies of anhydro-araIC 3'-acetate with the structurally related agents 2,2'-anhydro-1-(3'-O-acetyl-beta-D-arabinofuranosyl)cytosine (anhydro-araC 3'-acetate) and 2,2'-anhydro-1-(beta-D-arabinofuranosyl)-5-iodocytosine (anhydro-araIC). The presence of the 5-iodo substituent and/or the 3'-O-acetyl group on 2,2'-anhydro1-(beta-D-arabinofuranosyl)cytosine (anhydro-araC) did not alter the capacity of these agents to exert cytotoxic and antineoplastic activity against L1210, P388, L5178Y and human leukemia cells and against human colon and rectal carcinomas, as well as antiviral activity against herpes simplex virus Type 1. All of the compounds caused inhibition of [3H]thymidine incorporation into the DNA of L1210 cells in culture, with anhydro-araIC 3'-acetate being significantly less inhibitory than the other derivatives. Little or no interference with RNA and protein synthesis was produced by these pyrimidine nucleosides. Both anhydro-araIC 3'-acetate and anhydro-araIC were potent inhibitors of the activity of DNA polymerase alpha from the L1210 leukemia at the nucleoside level, while anhydro-araC 3'-acetate and anhydroaraC were non-inhibitory; none of the agents caused inactivation of DNA polymerase beta. The findings suggest that the antineoplastic and antiviral activities of the 2,2'-anhydro-arabinosylcytosine nucleosides may be the result of biochemical actions different from those of araC.