Sp3 is an ubiquitously expressed transcription factor, closely related to Sp1 but, unlike Sp1, it often functions as a transcriptional repressor. In this study we investigated the role of Sp3 in regulating the transcription of the human alpha2(I) collagen gene. We show that Sp1 and Sp3 specifically bind to three of the previously characterized cis -elements in this promoter, including two positive cis-elements between -303 and -271 and -128 and -123, and a repressor site between -164 and -159, but do not bind to the fourth cis-element bound by CBF. Functional analyses of Sp3 and Sp1 in Drosophila cells indicate that each protein transactivates the human alpha2(I) collagen promoter with equal potency and, when tested together, have an additive effect on the promoter activity. Furthermore, in vitro transcription assays demonstrate that both Sp1 and Sp3 are capable of supporting transcription from the collagen promoter independently of each other. However, when activities of both Sp1 and Sp3 are blocked with specific antibodies, in vitro transcription from this promoter is almost completely abolished. The results of this study demonstrate that Sp3 is as potent an activator of the human alpha2(I) collagen promoter as is Sp1 and that a transcriptional activity of the human alpha2(I) promoter is dependent on both proteins.