To gain an insight into the mechanisms of prostacyclin expression, a genomic DNA clone harboring 2.0 kb of the 5'-flanking sequence of the mouse prostacyclin synthase (PGIS) gene was isolated. The 5'-flanking region did not possess a TATA box, but contained a GC-rich region and several consensus cis DNA elements. The major product of the primer extension analysis suggested that the transcription of the gene started from 72 bases upstream of the translational initiation codon. To analyze the PGIS promoter activity, the 2.0 kb fragment was fused to the luciferase gene and transient transfection assays were conducted with cultured rat vascular smooth muscle cells (VSMC). The fragment showed significant promoter activity in the cells. Analysis of a series of 5'-deletion constructs showed that the 5'-flanking regions spanning bases -371 to -285 and -229 to -119 were important for the basal transcriptional activity of the mouse PGIS gene. Gel mobility shift assays revealed that DNA-protein complexes were formed with the nuclear extracts from VSMC, and that the formation of these complexes was inhibited by excess consensus Sp1 oligonucleotide. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in supershift of the band for the DNA-protein complex. In addition, mutation of two Sp1 recognition motifs residing at bases -297 to -289 and -197 to -192 markedly reduced the basal PGIS promoter activity and retarded the band in a gel mobility shift assay. These results indicated that binding of one Sp1 to two Sp1 sites on the promoter region activated the basal transcription of the PGIS gene.