3'Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) is a potent inhibitor of the replication of cultured L1210 cells, with an IC50 of 1 microM. When ribo- and deoxyribonucleosides were examined for their effects on 3'-NH2-dCyd-induced cytotoxicity, only dCyd could both prevent and reverse these effects. Furthermore, even when the maximum increase in modal cell volume was allowed to develop (24 hr) in the presence of 2.5 microM 3'-NH2-dCyd, the addition of 25 microM dCyd to the medium containing 3'-NH2-dCyd reduced the modal cell volume nearly to control levels within 24 hr. Examination of the viability of these cells by colony formation in soft agar, following as much as a 9.5-hr exposure of 1, 2.5 and 10 microM 3'-NH2-dCyd, revealed that the lethal effects of the 3'-NH2-dCyd treatment were not observed only when 25 microM dCyd was added to the medium during this time. However, the lethality of a 24-hr exposure of 2.5 and 10 microM 3'-NH2-dCyd could not be prevented either by removal of the drug from the medium or by a 24-hr exposure of the medium containing 3'-NH2-dCyd to 25 microM dCyd. When the effect of 3'-NH2-dCyd on DNA biosynthesis in L1210 cells was examined, it was found that radiolabeled dAdo incorporation decreased by approximately 60, 80 or 90% following a 2.5-hr exposure to 2.5, 10 or 20 microM 3'-NH2-dCyd respectively. The addition of 25 microM dCyd under the same conditions resulted in a greater amount of dAdo incorporation compared to the unrescued cultures. Deamination of 3'-NH2-dCyd by partially purified human cytidine-deoxycytidine deaminase was about 2.5% that of either Cyd or dCyd deamination. The deaminated derivative, 3'-amino-2',3'-dideoxyuridine, was significantly less cytotoxic even at 50 microM.