We have reported that glucocorticoids and cyclic nucleotides regulate tissue-type plasminogen activator activity in HTC rat hepatoma cells primarily by modulating plasminogen activator inhibitor (PAI-1) gene expression. To investigate the molecular mechanisms underlying this hormonal regulation, we have cloned the rat PAI-1 gene from an HTC genomic library. The gene is approximately 10.5 kilobases (kb) in size and is comprised of nine exons and eight introns. Comparison of this gene with the human PAI-1 gene (Bosma, P. J., van den Berg, E., Kooistra, T., Siemieniak, D. R., and Slightom, J. L. (1988) J. Biol. Chem. 263, 9129-9141) revealed strict conservation of the exon-intron structure. The start site of transcription is identical to that in the human PAI-1 gene. The complete nucleotide sequence of the rat PAI-1 gene, including 2.4 kb of 5'- and 1 kb of 3'-flanking DNA, has been determined. Two regions in the 5'-flanking sequence of the rat PAI-1 gene show a high degree of similarity to sequences in the upstream region of the human PAI-1 gene: a 60-base pair region from -90 to the TATA box (90% identical), and a more distal sequence located at -753 to -510 (greater than 80% identical). Both of these regions have been shown to be involved in the glucocorticoid regulation of expression of the human PAI-1 gene (van Zonneveld, A.-J., Curriden, S. A., and Loskutoff, D. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5525-5529), although neither region contains the hexamer consensus sequence for glucocorticoid response elements. Five putative glucocofticoid response elements, 100% identical to the hexamer consensus sequence, were identified in the 5'-flanking region of the rat gene, none of which is present in the regions of sequence similarity. Seven putative cyclic AMP response elements that are 75% identical to the octamer consensus sequence are located upstream of the rat gene. There is only one such sequence within the 5'-flanking region of the human gene; however, that sequence, at -61, is 100% conserved in the rat gene.