Genes that confer cellular resistance to cytotoxic agents have potential applications both for marrow protection following cancer chemotherapy and as dominant selectable markers for the selection of genetically modified hematopoietic cell populations in vivo. Cytidine analogues, including cytosine arabinoside (Ara-C), 2',2'-difluorodeoxycytidine (gemcitabine), and other drugs represent a clinically important class of chemotherapeutic agents for which no drug resistance gene has yet been described. Studies were performed to determine whether forced expression of a gene encoding the enzyme cytidine deaminase can confer resistance to cytosine arabinoside (Ara-C) and gemcitabine in vitro. A pooled population of NIH3T3 cells overexpressing cytidine deaminase from a retroviral construct based on the LXSN-vector (LCDSN) demonstrated a 4.5-fold increased resistance to Ara-C based on the 50% inhibitory concentration (ID50) and a 3.7-fold increased resistance to Ara-C based on the 80% inhibitory concentration (ID80) relative to cells transduced with a control vector. In the hemopoietic cell line CCRF-CEM, the same retroviral construct conferred a 2.1-fold increased resistance to Ara-C by ID50 and a 3.0-fold increased resistance to Ara-C by ID80 relative to cells transduced with a control vector. CEM cells transduced with LCDSN were also resistant to gemcitabine (2.4-fold by ID50 and 2.5-fold by ID80). Furthermore, Ara-C-resistance could be completely reversed in LCDSN-transduced CEM cells by a specific inhibitor of cytidine deaminase, tetrahydrouridine (THU). Expression of the transgene was confirmed by RNase-protection assay and by an enzyme activity assay. These results provide the first direct evidence that forced expression of cytidine deaminase confers cellular resistance to Ara-C and gemcitabine.