Arabinofuranosyl-5-azacytosine (ara-AC), a nucleoside combining the structural elements of 5-azacytidine and arabinofuranosylcytosine, exhibited unusually wide therapeutic activity against several murine leukemias and all three human xenografts of the National Cancer Institute tumor panel. Activity was observed following either a daily or an intermittent regimen of treatment in the i.p. L1210 model. However, when multiple doses were administered on each treatment day, a greater therapeutic effect was produced and the total dose was reduced. Extensive necrosis was observed by light and electron microscopy in P388 tumors treated with ara-AC. Following s.c. administration, ara-AC caused regression of the mammary and lung xenografts (MX-1 and LX-1) and a 93% inhibition of the human colon tumor (CX-1); other analogues of this drug failed to demonstrate a comparably broad spectrum of activity. Morphological assessment of treated xenografts revealed a general loss of cell-to-cell contact and abundant necrosis 24 h after the administration of ara-AC. In culture, the 50% inhibitory concentrations of ara-AC for P388 and L1210 cells at 24 h were 1.9 and 4.5 microM, respectively, and the decline in replication rates was dependent on drug concentration. The cytocidal nature of the drug was demonstrated by cloning experiments in which it was observed that ara-AC abolished the clonogenicity of lymphoblasts but was only minimally cytotoxic to normal murine bone marrow progenitor cells. As adjudged by flow cytometry, the drug induced a distinct slowing of cell cycle traverse through S phase. Induction of the differentiation of HL-60 cells in culture was another cytotropic effect of this drug. At 44% differentiation (10 microM ara-AC), 50% of the cultured cells were viable. Its broad spectrum antitumor activity, its selective toxicity to tumor cells, and its ability to produce cytodifferentiation render ara-AC of interest as a potential antineoplastic agent in humans.