Phosphorothioate oligodeoxynucleotides belong to a class of polyanions that bind to the third variable domain (v3) of HIV-1 gp 120 and inhibit infectivity of a wide variety of HIV-1 isolates. This potent v3 binding of phosphorothioate oligodeoxynucleotides, which is relatively independent of the nucleotide sequence of the oligodeoxynucleotides, decreases with chain length (below 18-mers) and is low for 8-mers. However, recent studies have observed a nucleotide sequence-dependent augmentation of phosphorothioate oligodeoxynucleotide binding to v3 for 8-mers that contain the S-dG₄ motif (e.g., SdT₂G₄T₂) and have suggested that formation of quadruple helical tetraplexes (G-tetrads) is associated with the acquisition of v3 binding ability by small phosphorothioate oligodeoxynucleotides. In the current study, a series of SdG₄-containing oligodeoxynucleotides were synthesized with varying tandem length (including the 8-mer SdT₂G₄T₂, the 12-mer SdG₄T₄G₄, and the 28-mer SdG₄(T₄G₄)3) and compared with phosphorothioate oligodeoxynucleotides (with similar lengths or related sequences) for (1) their inhibition of the binding of mAb 9284, which binds to the N-terminal portion of the v3 loop, (2) the values K_c when these compounds are used as competitors of the rgpl20-binding of an alkylating phosphodiester oligodeoxynucleotide probe, and (3) inhibition of HIV-1 infectivity in a cell-cell transmission model. The presence of S-dG₄ motifs and the number of tandem motifs augmented v3 binding and anti-HIV-1 infectivity for small (8-mer or 12-mer oligodeoxynucleotides) but did not significantly augment the potency of 28-mers. Whereas tetraplex formation of SdT₂G₄T₂ may contribute to its v3 binding, the 12-mer SdG₄T₄G₄ does not migrate as a tetraplex on nonreducing gels, suggesting that S-dG₄ motifs may augment anti-HIV activity by multiple mechanisms.