In vitro culture of H9 human lymphoid cells in the presence of 5.0 microM dideoxycytidine (ddC), for about 40-45 days, selected cells (H9-ddC cells), which were resistant to the drug and cross-resistant to AZT (zidovudine) and 5-fluoro-2'-deoxyuridine (FdUR). The major mechanism of cross-resistance to AZT and FdUR in these cells was low cellular activity of thymidine kinase (TK). To explore molecular mechanisms of the reduced TK activity in H9-ddC cells, the mRNA expression of TK1 and TK2 and western blot analysis of TK1 protein were performed. RT-PCR analysis revealed that in H9-ddC cells the expression of both TK1 and TK2 mRNA was reduced to 27.1% and 79.4%, respectively. The reduced TK1 gene expression was confirmed by an absence of a detectable TK1 protein band in western blot of H9-ddC cells. These results demonstrate that long-term treatment of H9 cells in the presence of ddC down-regulated TK1 and TK2 gene expression and reduced the expression and activity of TK in the resistant cells.