Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC50: >3000 nM), and the cladribine resistant CEM (IC50: 150 nM) and HL-60 (IC50: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC50; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.