To identify cis-elements regulating PMA-induced prostaglandin H synthase-1 (PGHS-1) gene expression in the human megakaryoblast cell line, MEG-01, we performed promoter reporter assays with a luciferase reporter vector containing the −2030/−22 region of the human PGHS-1 gene. PMA treatment for 24 h increased PGHS-1 promoter activity by twofold. Mutagenesis studies of the promoter revealed a single Sp1 site essential for PMA-inducible transcription. Insertion of a highly conserved 100 bp sequence cloned from intron 8 into the −2030/−22 reporter plasmid enhanced PMA-dependent transcription 10-fold. Mutation of either a consensus AP-1 site within intron 8 or the Sp1 site in the promoter reduced PMA-induced activity by 80–100%. Gel shift assays using the intron 8 AP-1 sequence demonstrated the formation of an AP-1-specific DNA–protein complex. Our results suggest that inducible PGHS-1 gene expression involves the coordinate functioning of a Sp1 site in the promoter and an AP-1 site in intron 8.