Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K_d = 14 μM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5′ ³²P-SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC₂₈, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate-treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media.