Type I collagen is a heterotrimeric protein composed of twoα1(I) and oneα2(I) polypeptide chains, each encoded by a unique gene. Both collagen genes are coordinately regulated in response to a variety of endogenous and exogenous stimuli. Collagen genes are mainly regulated by transcriptional mechanisms, although post-transcriptional regulatory mechanisms have occasionally been documented. To understand the molecular basis of transcriptional control, we have been studying thecis-regulatory sequence motifs of human Proα1(I) collagen gene andtrans-acting factors with which these elements interact. The major elements include TATA and CCAAT boxes, Ap-1, Ap-2, NF-1 and Sp1, and a unique TGFβ1-activating element. Transcription factor Sp1 is obligatory for the activation of the Proα1(I) promoter since it failed to be activated inDrosophila SL2 cells that lack Sp1. Of the six putative Sp1 motifs in the Proα1(I) collagen promoter and the first intron, the most proximal Sp1 element located at −87 to −82 bp was sufficient for its Sp1-dependent activation. Additionalcis-acting motifs in the intron of the Proα1(I) gene, including an Ap-1 site, participated in its regulation by TGFβ1 and okadaic acid. Thus, activation of Proα1(I) promoter involves combinatorial actions of multiple ubiquitous and uniquecis-regulatory elements.