2',2'-Difluorodeoxycytidine (gemcitabine, dFdCyd) is a deoxycytidine analogue with promising antitumor activity. In order to be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to dFCyd in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of dFdCyd. The IC50, defined as the concentration of dFdCyd causing 50% growth inhibition, at 72 h exposure increased from 0.6 nM dFdCyd in A2780 to 92 microM in the resistant variant, named AG6000. Although the resistant cell line is routinely cultured in 6 microM dFdCyd, the resistant phenotype can be maintained for at least 10 passages without dFdCyd. AG6000 is cross-resistant to other drug which require activation by dCK, such as 1-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. There was no specific dCK activity in extracts from AG600 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse-transcribed and PCR-amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs. Chromosome spreads from the cell lines showed no major differences between A2780 and AG6000. The latter cell line was also cross-resistant to 2',2'-difluorodeoxyurdine, the deamination product of dFdCyd. Additionally, cross-resistance to the multidrug resistant drugs doxorubicin and vincristine was observed. This was not associated with the induction of P-glycoprotein, as determined by the RNase protection assay. Injection of AG6000 cells s.c. into nude mice demonstrated that the cell line had retained its tumorigenicity; AG6000 xenografts were not sensitive to dFdCyd treatment, in contrast to the parental A2780 tumors. No dFdCyd triphosphate accumulation was found in the resistant tumors, in contrast to the parental A2780 tumors. These results indicate that the dFdCyd resistance phenotype is stable, and mainly due to dCK deficiency.