The effects of 5-aza-2′-deoxycytidine (aza-dCyd) and 5,6-dihydro-5-azacytidine (H₂-aza-Cyd) on the integrity of DNA from several mammalian cell lines were compared using the alkaline elution technique. While both compounds have been shown to inhibit DNA methylation, a direct comparison of their effects on DNA structure has not previously been reported. Exposure of L1210 cells to H₂-aza-Cyd (1–100 µg/ml) and simultaneous labeling with [¹⁴C]thymidine for 24 h resulted in the production of single-strand breaks in DNA, which were significantly repaired when cells were incubated in drug-free medium for an additional 24 h. This differed from our previous findings for aza-dCyd, confirmed here in parallel experiments, which showed that this compound produces alkali-labile lesions that persist for 48 h. The DNA effects of both drugs were significantly reduced when cells were prelabeled with [¹⁴C]thymidine, indicating that production of DNA lesions requires incorporation of the anomalous base. Studies utilizing pulse-labeled DNA indicated that aza-dCyd has little effect on the rate of DNA elongation, whereas H₂-aza-Cyd produced a complete inhibition for at least 6 h after drug removal. The contrasting pattern of DNA damage induced by these compounds in L1210 was also observed in two human lymphoblastoid cell lines, one of which was derived from a patient with xeroderma pigmentosum. We had previously concluded that alkali-labile sites in DNA from aza-dCyd-treated cells probably arise due to the chemical instability of aza-dCyd. In contrast, incorporated H₂-aza-Cyd is chemically stable. The singlestrand breaks produced in H₂-aza-Cyd treated cells were not of the alkalilabile type, and may represent an accumulation of DNA replication fragments and/or intermediates in an excision repair process. Thus, the DNA lesions produced by the two drugs have markedly different characteristics, and H₂-aza-Cyd should not be considered to be merely a stable pharmacological congener of aza-dCyd.