2-Chloro-2'-deoxyadenosine, (CldAdo) resistance was developed in the W1L2 human B lymphoblastoid (resistance factor, 160) and L1210 murine leukemia (resistance factor, 605) cell lines by continuous exposure to CldAdo. Cross-resistance studies showed that while the variant lines generally retained sensitivities to 9-beta-D-arabinofuranosyladenine (in the presence of 2'-deoxycoformycin), hydroxyurea, and Adriamycin, both were highly cross-resistant to 1-beta-D-arabinofuranosylcytosine (ara-C), 2', 2'-difluorodeoxycytidine, and 9-beta-D-arabinofuranosyl-2-fluoroadenine. Measurement of both phosphorylating and degrading enzyme activities demonstrated that initial phosphorylation of CldAdo and 2'-deoxycytidine were severely impaired in cell extracts from the resistant lines, whereas adenosine kinase activity remained unaffected and there was no apparent increase in cytoplasmic deoxynucleotidase activity using dCMP as substrate. Since previous reports indicated that either overexpression of Bcl-2 protein following bcl-2 transfection into cells resulted in, or high dCTP pools contributed to, ara-C resistance in experimental cell models, both of these parameters were assessed and found not to contribute to CldAdo resistance in the murine leukemia and human B lymphoblastoid cells. These studies show that a deficiency of 2'-deoxycytidine kinase activity is a major determinant of CldAdo acquired resistance in both the murine and human lymphoid lines.