In vivo transient expression and in vitro transcription experiments indicated that a segment between -170 and -40 bp upstream of the start of transcription of the mouse proalpha2(I) collagen gene was essential to activate transcription. DNase I protection experiments identified three strong footprints in this segment. Experiments with deletion mutants encompassing the sequences defined by these three footprints indicated that each of the three elements contributed to the transcriptional activity of the promoter. All three elements are GC-rich, redundant sites for a complex set of DNA binding proteins that includes SP1, other proteins that bind to an SP1 consensus site and proteins that bind to a Krox consensus site. In addition, the segment corresponding to the most proximal footprint also binds the multimeric CCAAT binding protein CBF. Addition of an excess amount of oligo- nucleotides corresponding to either of the two distal footprints significantly inhibited in vitro transcription of the -350 bp proalpha2(I) collagen promoter. Anti-SP1 antibodies that completely inhibited transcription of the early SV40 promoter had little effect on transcription of the wild-type -350 bp promoter, suggesting that SP1 has only a minor role in activity of this promoter. Our results show that the segment between base pairs -170 and -40 of the proalpha2(I) collagen promoter, which contains redundant binding sites for a complex set of nuclear proteins, is essential in the transcriptional activity of this promoter in fibroblasts.