The human dihydrofolate reductase (DHFR) promoter sequence contains two consensus binding sites for the Sp1 regulatory protein. We have determined the effect of intermolecular triplex DNA formation on Sp1 binding to the DHFR promoter. The DHFR Sp1 binding site I (-39 to -48 relative to the DHFR transcription start site) demonstrates concentration-dependent triplex formation with a 19-base pair G-rich oligonucleotide (GR19) which is complementary to the polypyrimidine strand. DNase I footprint analysis demonstrates that GR19 forms a DNA triplex structure with the DHFR promoter fragment in a sequence-specific manner. DNase I footprinting analysis also indicates that the orientation of binding of these G-rich oligonucleotides is antiparallel. CR19, a C-rich complementary oligonucleotide, on the other hand, does not form triplex. The DNase I protection pattern of DHFR promoter fragment incubated with both recombinant Sp1 and triplex-forming oligonucleotide suggests that triplex formation prevents Sp1 binding. This is confirmed by gel shift analysis which demonstrates that triplex formation by the Sp1 binding sequences of the DHFR promoter prevents recombinant Sp1 binding in a concentration-dependent manner. These results demonstrate that intermolecular triplex formation prevents regulatory protein binding in a sequence-specific manner.