Previous studies suggest that there may be species differences in the utilization of cis-elements of the type I collagen genes. The present study was designed to examine this possibility by focusing on two regions of the pro␣1(I) collagen promoter. One is the GC-rich A1 region (Ϫ194/168) that modulates transcriptional activity of the mouse promoter. The other contains a glucocorticoid response element (GRE) implicated in negative glucocorticoid regulation of the rat promoter. Unlike mouse A1 probes, probes representing the analogous human (Ϫ195/Ϫ168) and rat (Ϫ193/Ϫ179) regions failed to bind nuclear proteins in gel shift assays. Binding assays with mouse A1 probes containing base substitutions indicated that this behavior could be ascribed to five bases in the human, and two in the rat sequences. In addition, the pattern of expression of c-Krox, a protein that alters transcriptional activity via the mouse A1 element, differed in mouse and human tissues. Computer analysis revealed differences in the arrangement of GRE half-sites in human and rat pro␣1(I) collagen promoters. In a region of the human promoter (Ϫ700/673) analogous to the rat (Ϫ672/Ϫ633), there are three half-sites, each separated by two nucleotides, that cooperate in binding of glucocorticoid receptor. There also is a proximal half-site at position Ϫ335 of the human promoter that binds glucocorticoid receptor, but it is not present in the rat promoter. This study has defined several species-specific differences in the sequences and nuclear protein binding activity of regions involved in transcriptional activity of the pro␣1(I) collagen promoter. The results suggest that the A1 regions of the human and rat promoters examined here are unlikely to function as regulatory cis-elements, and they provide a framework for investigating the role of GREs in transcriptional regulation. They also suggest that species differences in cis-elements and transcription factors should be taken into consideration when using heterologous systems to study collagen gene regulation.