BACKGROUND: The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients. METHODOLOGY/PRINCIPAL FINDINGS: A hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells. CONCLUSIONS/SIGNIFICANCE: Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.