To be able to elucidate the function of cyclin D1 in the control of cell cycle progression and its role as an oncogene in tumorigenesis, it is of paramount importance to understand the mechanisms involved in the regulation of its expression. In the present study, we have cloned the human cyclin D1 gene and analysed the structure and function of 3kb of its 5'-flanking region. Several regulatory regions involved in both basal level and serum-induced expression were identified, two of which turned out to be of particular interest. One of these regions is involved in serum induction and is located 848-944 bp upstream of the initiation site. In agreement with this result, in vivo footprinting revealed a novel, strongly inducible protein binding site around positions-928 to-921. A second constitutively occupied binding site was mapped to a potential CRE at position-52. Cotransfection experiments showed that the cyclin D1 promoter is inducible by c-Jun, and that this induction is mediated predominantly through the protected putative CRE at-52.