We have found that chronically HIV-1(IIIB)-infected H9 cells showed 21-fold resistance to 1-beta-D-arabinofuranosylcytosine (ARA-C) compared with uninfected H9 cells. In the infected H9 cells, a 37% increase of dCTP pool and a 34% increase of dATP were observed, and no alteration of dTTP and dGTP was observed, compared with the uninfected H9 cells. A marked decrease of ARA-CTP generation was observed in the infected H9 cells after 3-h incubation with 0.1-10 microM ARA-C. The level of deoxycytidine kinase activity with ARA-C as substrate was similar in both the infected and the uninfected cells; however, a 37-fold increase of cytidine deaminase activity was observed in the infected H9 cells. These results indicate that the induction of cytidine deaminase activity by HIV-1(IIIB) infection conferred ARA-C resistance to H9 cells. This conclusion was supported by the observation that a marked reversal of ARA-C resistance in the infected H9 cells occurred after treatment with the inhibitor of cytidine deaminase, 3,4,5,6-tetrahydrouridine. The understanding of these cellular alterations in drug sensitivity may facilitate the development of effective therapeutic strategies against HIV-1-infected cells.