Chronic lymphocytic leukemia (CLL) is most commonly treated with the alkylating agent chlorambucil (CLB), although the nucleoside analogs, fludarabine (Flu) and 2-chlorodeoxyadenosine (CdA), are also effective in this disease. In this study, we investigated the in vitro cytotoxicity of CdA and CLB in CLL cells from 12 patients in vitro. Treatment with CLB for 6 h, followed by CdA for 18 h, resulted in 2.3- to 7.5-fold synergistic cytotoxicity in leukemic cells from 10 patients and an additive effect in cells from two patients. In general, synergy was greatest in patients who were sensitive to CLB or CdA, and could be enhanced by increasing the concentrations of CLB or CdA. Synergy was only observed if the cells were treated with CLB prior to CdA. Synergy could not be explained by an increase in the incorporation of CdA into DNA, or by the inhibition of repair of CLB-induced DNA crosslinks by CdA. In contrast to CLL cells, treatment of human marrow in vitro with CLB and CdA resulted in a low level of synergy for CFU-GM cells, and additive cell kill in erythroid progenitors. Thus, treatment with CdA and CLB can produce selective synergistic cell kill in CLL cells, and combination therapy may improve the therapeutic index of these agents in chemosensitive patients.