Transcriptional activation of p53-regulated genes is initiated by sequence-specific DNA binding of p53 to target binding sites. Regulation of sequence-specific DNA binding is complex and occurs at various levels. We demonstrate that DNA topology is an important parameter for regulating the selective and highly specific interaction of p53 with its target binding sites. Specific binding of wild-type p53 is greatly enhanced when cognate binding sites are present in a non-linear stem-loop conformation. The C-terminal domain plays a key role in regulating the specific interactions of p53 with target binding sites in a DNA conformation-dependent manner. The C-terminal domain is required for binding to target sites in a non-linear DNA conformation in contrast to the strong inhibitory effects of the C terminus on p53 interaction with linear DNA. We propose that selective binding of p53 to various promoters may be determined by the DNA conformation within p53 cognate sites.