The nucleoside analog 2',3'-dideoxycytidine (ddCyd) has been shown to inhibit the infectivity and cytopathic effect of human immunodeficiency virus on human OKT4+ lymphocytes in vitro. Metabolism of ddCyd by human T-lymphoblastic cells (Molt 4) negative for human immunodeficiency virus and OKT4 was examined. Molt 4 cells accumulated ddCyd and its phosphorylated derivatives into acid-soluble and acid-insoluble material in a dose-dependent manner. For each concentration tested, 2',3'-dideoxycytidine triphosphate represented 40% of the total acid-soluble pool of ddCyd metabolites. Uptake of 5 microM ddCyd was linear for 4 h after addition of drug. Efflux of ddCyd metabolites from cells followed a biphasic course with an initial retention half-life of 2.6 h for 2',3'-dideoxycytidine triphosphate. DNA, but not RNA, of cells incubated with [3H]ddCyd became radiolabeled. Nuclease and phosphatase treatment of DNA followed by reverse-phase high pressure liquid chromatography showed that the nucleoside was incorporated into DNA in its original form. ddCyd was not susceptible to deamination by human Cyd-dCyd deaminase. It was a poor substrate for human cytoplasmic and mitochondrial dCyd kinases, with Km values of 180 +/- 30 and 120 +/- 20 microM, respectively. DNA polymerases alpha, beta, and gamma varied in their sensitivity to inhibition by ddCTP with Ki values of 110 +/- 40, 2.6 +/- 0.3, and 0.016 +/- 0.008 microM, respectively; however, inhibition was competitive with dCTP in each case.