Continuous in vitro cultivation of human lymphoid H9 cells in the presence of 0.5microM arabinosyl-cytosine (araC) resulted in cell variant, H9-araC cells, that was >600-fold resistant to the drug and cross resistant to its analogs and other unrelated nucleosides, e.g. dideoxycytidine (5-fold), thiacytidine (2-fold), 2-fluoro-adenine arabinoside (8.3-fold), and 2-chloro-deoxyadenosine (2.1-fold). Compared to the parental cell line, the resistant cells accumulated <1% araCTP, and had reduced deoxycytidine kinase (dCK) activity (31.4%) and equilibrative nucleoside transporter 1 (ENT1) protein. The expression of the dCK gene in araC resistant cells was reduced to 60% of H9 cells, which correlated with lower dCK protein and activity. Whereas, there was no difference in the expression of ENT1 mRNA between the cell lines, ENT1 protein content was much lower in the resistant cells than in H9 cells. The concentrative nucleoside transporter (CNT3) was slightly increased in H9-araC cells, but CNT2, and MDR1 remained unaffected. Although a definitive correlation remains to be established, the amount of Sp1 protein, a transcription factor, that regulates the expressions of dCK, nucleoside transporters and other cellular proteins, was found reduced in H9-araC cells. Like ENT1, the Sp1 mRNA levels remained unaffected in H9-araC whereas protein contents were reduced. These observations are indicative of differences in the production and/or turnover of ENT1 and Sp1 proteins in H9-araC cells. Since nucleoside transporters and dCK play an important role in the activity of potential antiviral and anticancer deoxynucleoside analogs, understanding of their regulation is important. These studies show that the exposure of cells to araC, in vitro, is capable of simultaneously affecting more than one target site to confer resistance. The importance of this observation in the clinical use of araC remains to be determined.