We previously demonstrated that a segment of the human alpha1 type I procollagen gene (COL1A1) promoter encompassing nt -174 to -84 is responsible for the highest transcriptional activity in collagen producing cells in vitro. Here, we identified two almost identical tandem NF-1/Sp1 binding sites located between nt -129 to -107 (distal element) and nt -104 to -77 (proximal element) that are responsible for the basal regulation of COL1A1 transcription in normal human dermal fibroblasts. Transient transfection studies revealed that 85% of the basal COL1A1 promoter activity resides within the distal element; however, site-directed mutagenesis within the CCAAT motif in the proximal element resulted in a 98% decrease of the COL1A1 promoter activity. We conclude that each of the NF-1/Sp1 tandem binding sites has a different function. The distal element drives the transcriptional activity of the COL1A1 promoter but is not sufficient for its basal expression, whereas the NF-1 binding site in the proximal element is essential for in vitro COL1A1 gene transcription.