A very long chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9), was detected in an eicosapentaenoic acid (EPA)-producing marine bacterium, which was isolated from the mid-latitude seashore of Hokkaido, Japan, and was tentatively identified as mesophilic Shewanella sp. strain osh08 from 16S rRNA gene sequencing. The geometry and position of the double bonds in this compound were determined physicochemically to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25, and 28. Although C31:9 was detected in all of the seven EPA-or/and docosahexaenoic acid-producing bacteria tested, an EPA-deficient mutant (strain IK-18) of one of these bacteria had no C31:9. Strain IK-18 had defects in the pfaD gene, one of the five pfa genes responsible for the biosynthesis of EPA.Although Escherichia coli DH5 does not produce EPA or DHA inherently, cells transformed with the pfa genes responsible for the biosynthesis of EPA and DHA produced EPA and DHA, respectively, but not C31:9. These results suggest that the Pfa protein complex is involved in the biosynthesis of C31:9 and that pfa genes must not be the only genes responsible for the formation of C31:9. In this report, we determined for the first time the molecular structure of the C31:9 and discuss the possible biosynthetic pathways of this compound. Key words