AbstractTranscriptional regulation of the human α 2(I) procollagen proximal promoter involves the interaction of trans‐acting factors at the inverted CCAAT box (G/CBE) located at position ‐ 80 and an adjacent GGAGGCCC‐box at ‐ 70. Both these elements have previously been shown to be essential for activity of the human promoter. This study investigated nucleotide differences at three sites (‐74, ‐ 72 and ‐ 71) between the human and mouse promoters that were sufficient to abolish trans‐acting factor binding with the mouse sequence (GGAGACGT). Two distinct DNA‐protein interactions were detected on the human ‐ 107/ + 54 promoter fragment while a single interaction was observed at the equivalent mouse promoter. One of these factors is the CCAAT‐binding factor (CBF) and it's binding was observed on both the human and mouse promoters. Although the GGAGGCCC DNA‐binding element was not detected on the mouse promoter, GGAGGCC‐binding proteins were present in mouse nuclear extracts as observed by their interaction with the human promoter. Functional analysis of the human and mouse ‐ 343/ + 54 and ‐ 107/+ 54 promoter regions revealed significant differences between species; the human constructs having higher activity than the mouse. The differences in promoter activity between species may in part be a result of the nucleotide differences in the GGAGGCCC‐box. Mutations in this region of the human ‐ 107/+ 54 promoter prevented DNA‐protein interaction and lowered promoter activity. These results support the hypothesis that the GGAGGCCC‐box in the human α 2(1) procollagen promoter has a regulatory function and that there exists a species‐specific difference in transcription factor binding and regulation of the gene. IUBMB Life, 57: 363‐370, 2005