Recent studies on the regulation of protein C gene transcription revealed the presence of three transcription-factor binding sites in the close proximity to the transcription start site. The proximal 40 bp upstream of the transcription-initiation site contain two, partly overlapping, binding sites for the liver-enriched hepatocyte nuclear factor (HNF)-3 and one binding site to which HNF-1 and HNF-6 bind in a mutually exclusive manner. In order to examine the functionality of the tight alignment of transcription-factor binding sites around the transcription-initiation site, we performed insertional mutagenesis experiments. Sequences were inserted at position -21, separating both HNF-3 binding sites from the HNF-1-HNF-6 binding site, and position -5, separating the HNF-3-HNF-1-HNF-6 complex from the transcription start site. All insertions were made in the context of the protein C gene -386/+107 promoter region and tested for activity by transient transfection experiments. Insertions at position -21 resulted in a combined distance- and DNA-turn-dependent increase in protein C gene expression. Insertions of variable length at position -5 decreased protein C gene expression in a DNA-turn-dependent manner. However, this turn-dependent decrease was accompanied by a distance-dependent increase in promoter activity. This is the first report in which changing the spacing between adjacent transcription-factor binding sites results in enhanced transcription, indicating the submaximal alignment of promoter elements in the wild-type protein C gene promoter region.