We have evaluated the specificity and the variation in activity against human immunodeficiency virus (HIV) infection of antisense oligodeoxynucleotides (ODNs) with regard to factors such as dose-response range, number and choice of experimental controls, backbone modifications of the ODNs, type of cell infection, length of assays, and delivery approach. The highest level of inhibition was achieved in our long-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB) and treated with free phosphorothioate-modified ODNs (PS-ODNs). The highest level of specificity was observed in our short-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB) and treated with free PS-ODNs. The highest potency (IC50 level) was observed in our short-term chronic-infection model with (DLS)-delivered ODNs in which the DLS delivery improved the ODN activity up to 106 times compared to the activity of free ODNs. Thus, the near blocking of HIV replication obtained when using PS-ODNs appears because of the addition of extracellular and/or membrane effects. The higher efficacy of PS-ODNs compared to unmodified ODNs, when both are delivered with the DLS system, was demonstrated solely in our short-term assay with MOLT-3 cells. Important variations in the level of sequence specificity were observed and depended on the type of control used and the type of cell assay employed. It seems that all 3 groups of control-tested, random, sense sequence, and non-antisense T30177 ODNs might have distinct activity and, consequently, different modes of action in inhibiting HIV replication. Our data buttress the notion that the contribution of the sequence-specific mediated mode of action is minor compared to the other mechanisms involved in ODN antiviral activity.