The cDNA encoding prostacyclin synthase (PGIS) was isolated from a bovine arota cDNA library. The cDNA contained an open reading frame of 1500 nucleotides encoding a polypeptide of 500 amino acids with a Mr of 56,675. The predicted amino acid sequence contains four polypeptide sequences determined from purified bovine PGIS. Comparison of the PGIS sequence with other protein sequences in protein data banks indicates that PGIS has considerable sequence similarity with cytochrome P450s; the closest related sequence is that of human cholesterol 7-α-monooxygenase (CYP 7). The PGIS sequence is consistent with several structural elements found in other P450s, including a putative membrane anchoring segement, a helix I which forms an α-helix backbone through the center of the enzyme and a heme-binding pocket. Overall, the PGIS has ≤ 31 % identity to other P450s, suggesting that PGIS represents a previously undefined family of cytochrome P450. PGIS shares only 26 % sequence identity with human thromboxane synthase and has a different hydropathy pattern near the amino terminus, suggesting a different membrane anchoring topology. Availability of PGIS cDNA will allow us to elucidate the different catalytic mechanisms between these two enzymes.