Gemcitabine, a novel pyrimidine nucleoside antimetabolite, has shown clinical antitumor activity against several tumors (breast, small-cell and non-small-cell lung, bladder, pancreatic, and ovarian). We have developed a drug-resistance profile for gemcitabine using eight drug-resistant P388 leukemias in order to identify potentially useful guides for patient selection for further clinical trials of gemcitabine and possible noncrossresistant drug combinations with gemcitabine. Multidrug-resistant P388 leukemias (leukemias resistant to doxorubicin or etoposide) exhibited no crossresistance to gemcitabine. Leukemias resistant to vincristine (not multidrug resistant), cyclophosphamide, melphalan, cisplatin, and methotrexate were also not crossresistant to gemcitabine. Only the leukemia resistant to 1-beta-D-arabinofuranosylcytosine was crossresistant to gemcitabine. The results suggest that (1) it may be important to exclude or to monitor with extra care patients who have previously been treated with 1-beta-D-arabinofuranosylcytosine and (2) the lack of crossresistance seen with gemcitabine may contribute to therapeutic synergism when gemcitabine is combined with other agents.