Oligonucleotide compounds composed of only deoxyguanosine and deoxythymidine were able to significantly inhibit human immunodeficiency virus type −1 (HIV-1)–induced syncytium formation and virus production (as measured by p24 core antigen expression) in an acute infection assay system. The oligonucleotides did not share any homology with or possess any complementary (antisense) sequence motifs to the HIV-1 genome. The guanosine/thymidine–containing oligonucleotides (GTOs) that showed this anti-HIV activity contained natural phosphodiester (PD) linkages (backbones) between the nucleosides. One of the PD oligonucleotide sequence motifs tested was capable of inhibiting HIV-1-induced syncytium formation and p24 production with a median effective dose in culture (ED50) in the submicromolar range. In addition, oligonucleotides tested were able to significantly suppress HIV-1 p24 levels ≥7 days after removal of the drug from the infected cell culture medium. The growth inhibition properties (toxicity) of this genre of oligonucleotides was determined to be well above the ED50values yielding high selective indexes. In vitro results showed that GTOs with PD backbones were potent competitive inhibitors of HIV-1 reverse transcriptase. These same molecules were capable of blocking the interaction between gp120 and CD4. All measured activities of these molecules were increased by factors of 10–500 when the PD backbone was replaced with a PT backbone in a sequence-dependent manner. The enhanced antiviral activity displayed by the sulfur group on the oligonucleotide backbone and the lack of any sequence-specific interactions suggest that a percentage of antiviral activity of oligonucleotide-based therapeutics is due to mechanisms other than those originally postulated for oligonucleotides. The good selective index of GTOs coupled with the prolonged suppression of HIV-1 in culture after removal of oligonucleotides from the infected cell culture make this a class of compounds that warrant investigation as therapeutic agents to be used against HIV-1.