3'-Amino-2',3'-dideoxycytidine (3'-NH2-ddCyd) is a 3'-modified deoxycytidine analog that specifically inhibits DNA synthesis. Inhibition of chain elongation at the replication fork was examined utilizing a batch hydroxylapatite chromatography method. Exponentially growing cells were exposed to 3'-NH2-ddCyd and the diterpene aphidicolin for 9.5 hr at concentrations that inhibited DNA synthesis by approximately 60 and 90%, as determined by precursor uptake. Both agents demonstrated a concentration-dependent inhibition of pulse labeling of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) generated by a limited alkaline lysis procedure. Upon removal of drug, the rate of elongation of pulse-labeled DNA was similar to that of untreated cells at both concentrations of aphidicolin and at the low concentration of the amino analog. Under these conditions, no reduction in cell survival was observed using the clonogenic assay technique. However, at the high concentration of 3'-NH2-ddCyd, the rate of elongation following drug removal was one-third that of untreated cultures, and a 50% loss in cell viability was observed. Furthermore, upon incubation of purified dsDNA with the Klenow fragment of Escherichia coli DNA polymerase I or purified ssDNA with calf thymus terminal deoxynucleotidyl transferase, only DNA from cells treated with the high concentration of 3'-NH2-ddCyd served as a poor template for further synthesis. The results indicate that 3'-NH2-ddCyd, in a concentration-dependent manner, inhibits DNA synthesis by reducing the rate of chain elongation at the replication fork, which subsequently leads to a functional blocking of 3'-ends in DNA. The data suggest that there may be a relationship between loss of cell viability and reduction in the number of 3'-ends available for DNA replication.