The nucleoside analogs fludarabine and gemcitabine inhibit cellular DNA synthesis by two different mechanisms: (1) direct termination of DNA strand elongation after the triphosphate of each drug is incorporation into DNA; and (2) indirect inhibition of DNA synthesis by decreasing cellular dNTPs through inhibition of ribonucleotide reductase. The present study demonstrated that incorporation of the analogs into DNA is critical for the cytotoxic action of these drugs in human T lymphoblastoid CEM cells. S phase cells, which actively incorporated the analogs into DNA, were most sensitive to the cytotoxic action of these compounds. A relatively short-term (5-24 h) cessation of cellular DNA synthesis without analog incorporation was not sufficient to cause cell death. The drug-treated cells died through apoptosis characterized by generation of internucleosomal DNA fragmentation and apoptotic morphology. Induction of high molecular mass (50-500 kb) DNA fragmentation was also observed in cells undergoing apoptosis; this type of DNA degradation was strongly correlated with the analog-induced cell death process. Inhibition of the analog incorporation into DNA by aphidicolin blocked both types of DNA fragmentation and apoptotic morphology, indicating the essential role of analog incorporation into DNA in drug-induced cell death.