Two species of human thromboxane synthase (TXS) cDNA, called TXS-I and -II, were previously isolated (K. Ohashi, K.-H. Ruan, R. J. Kulmacz, K. K. Wu, and L.-H. Wang, 1992, J. Biol. Chem. 267, 789-793). TXS-II differs from TXS-I by a 163-bp deletion near the 3'-end of the coding region. Both types of TXS mRNA have now been demonstrated to be present in various blood and lung cultured cells. Analysis of the exon-intron boundaries of TXS genomic DNA revealed that the two mRNAs are generated via alternate splicing: TXS-II is produced by skipping an entire 163-bp exon which encodes the polypeptide segment containing the heme-binding cysteine conserved among other P450s. The mechanism by which alternate splicing occurs is probably due to the presence of a more powerful potential as the 3' acceptor site in the intron following the 163-bp exon. When expressed in baculovirus system, recombinant TXS-I catalyzed the formation of thromboxane A2 and 12-hydroxyheptadecatrienoic acid (HHT), whereas recombinant TXS-II did not synthesize thromboxane A2 or HHT. Alternate splicing of TXS RNA transcript thus may provide a mechanism for limiting cellular biosynthesis of thromboxane A2.