The cytosolic 3'-to-5' exonuclease from chronic lymphocytic leukemia cells was highly purified, and its ability to remove beta-D- and beta-L-nucleotide analogs from the 3'-end of DNA was determined. The relative rate of excision of beta-D-ddCMP, beta-L-ddCMP, beta-L-FddCMP, beta-L-SddCMP, beta-L-Fd4CMP, and beta-L-OddCMP from the 3'-end of a single-stranded oligonucleotide primer or a primer annealed with complementary DNA and/or RNA templates was assessed. The rate of excision of beta-D-nucleotides from the 3'-end of DNA was higher than that of beta-L-nucleotides, which could be partly attributable to the affinity of the enzyme to beta-D-nucleotide-terminated DNA being 5-fold higher compared with that of beta-L-nucleotide-terminated DNA. The rate of removal of beta-L-Fd4CMP and beta-L-OddCMP from the 3'-end of DNA was at least 8 to 10 times lower compared with that of beta-L-SddCMP. HIV reverse transcriptase could elongate DNA primers after the removal of chain terminators by the cytosolic exonuclease. Concentrations of nucleoside 5'-monophosphate analogs that inhibit the cytosolic exonuclease by 50% were estimated. Among the nucleoside 5'-monophosphate analogs examined, beta-L-Fd4CMP appeared to be the most effective inhibitor of the cytosolic exonuclease, with an ID(50) value of 38 microM.