Levels of expression of the platelet-derived growth factor (PDGF) A-chain gene differ significantly in normal and transformed cells. We have identified an S1 nuclease-sensitive site in a GC box located at -55 to -72 in the PDGF A-chain promoter region. We now demonstrate that a 24-base, G-rich complementary oligonucleotide anneals specifically to the C-rich strand of the GC box and protects the GC box from nicking by S1 nuclease whereas the C-rich complementary oligonucleotide and its double-stranded counterpart do not. In transient transfection assays, expression of the PDGF A-chain gene is sharply reduced by deletions within the GC box or if the 24-base G-rich complementary oligonucleotide is preincubated with the promoter construct prior to transfection. The data suggest that the GC box of the PDGF A-chain gene may promote a non-B-form DNA structure, which is recognized by S1 nuclease and which anneals to a short complementary G-rich oligonucleotide. The data also suggest that this non-B-form DNA is important for efficient transcription of PDGF A-chain gene.