Gemcitabine (2'-deoxy-2',2'-difluorocytidine; dFdC) is a novel nucleoside analog that has shown clinical activity against solid tumors. The semiautomated fluorometric microculture cytotoxicity assay (FMCA) was used for evaluation of the cytotoxicity of gemcitabine in vitro in primary cultures of human cells from patients with hematologic or solid tumors. The activity pattern of cytarabine (ara-C), 2-chlorodeoxyadenosine (CdA), etoposide (VP-16), doxorubicin, and cisplatin were included for comparison. One hundred forty samples were tested using continuous drug exposure. Gemcitabine showed high activity against hematologic samples, whereas little or no activity was observed in the solid tumor groups. A similar pattern of activity also was observed for ara-C and CdA, whereas etoposide, cisplatin, and doxorubicin were relatively more active in solid tumors. Cross-resistance analysis between gemcitabine and the standard drugs revealed the following rank order of correlation coefficients: ara-C > doxorubicin > CdA > cisplatin > VP-16. The results indicate that gemcitabine is differentially active against hematologic tumors and that the activity pattern of gemcitabine resembles that of ara-C. However, these results also indicate that gemcitabine may be more active against some solid tumor groups in comparison to ara-C and CdA.