2',2'-Difluorodeoxycytidine (gemcitabine; dFdC) is a nucleoside analog with promising antitumor activity. To be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to gemcitabine in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of gemcitabine. At 72 hours' exposure the IC50, defined as the concentration of gemcitabine causing 50% growth inhibition, increased from 0.6 nmol/L gemcitabine in A2780 to 92 mumol/L in the resistant variant, AG6000. AG6000 is cross-resistant to other drugs that require activation by dCK, such as I-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. AG6000 was also cross-resistant to 2',2'-difluorodeoxyuridine (dFdU), the deamination product of gemcitabine. In addition, cross-resistance to the multidrug-resistance drugs doxorubicin and vincristine was observed. This was not associated with induction of P-glycoprotein. No accumulation of gemcitabine triphosphate could be detected in AG6000 cells, in contrast to the parental A2780 cells. There was no specific dCK activity in extracts from AG6000 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse transcribed and polymerase chain reaction 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. Although the resistant cell line is routinely cultured in 6 mumol/L gemcitabine, the resistant phenotype can be maintained for at least 10 passages without gemcitabine. These results indicate that the gemcitabine resistance phenotype is stable and mainly due to dCK deficiency.