The 3500-base pair region located immediately upstream of the transcriptional start site of the human pro-alpha 2(I) collagen gene contains all the sequences necessary for cell-specific transcription. In transient expression assays, the pro-alpha 2(I) collagen promoter directed the production of high levels of bacterial chloramphenicol acetyltransferase in collagen-producing human fetal fibroblasts. Enzyme activity, on the other hand, was nearly undetectable in extracts from collagen-nonproducing immortalized lymphoblasts. Deletion experiments narrowed the active segment of the human promoter to a phylogenetically conserved sequence comprised between nucleotides-376 and -108, relative to the initiation site of transcription. In similar analyses, the pro-alpha 1(I) collagen gene failed to direct cell-specific transcription. As part of this study, the controversial issue surrounding the putative enhancer element in the first intron of the human pro-alpha 1(I) collagen gene also has been reconsidered. Accordingly, we now propose a more restricted definition of this cis-acting DNA element since its action is exerted in an orientation-preferred manner and with a strong specificity for its own promoter. Moreover, stimulation does not appear to be tissue-specific. Finally, evidence is presented supporting the notion that although structurally different and distinctly arranged, the regulatory sequences of the type I collagen genes may bind similar trans-acting factors.