Antisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have reexamined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3' sequences of the ODNs determine their effect. At concentrations of 10 micromol/L (for phosphorothioate ODNs) or 25 micromol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3' positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5' monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2'deoxycytidine 5'-monophosphate] is not cytotoxic), on their concentration (d-GMP [2'-deoxyguanosine 5'-monophosphate], TMP [thymidine 5'-monophosphate], and d-AMP [2'-deoxyadenosine 5'-monophosphate] are cytotoxic at concentrations between 5 and 10 micromol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3' hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.