cis-3,4-Methylene hexanedioic acid has been discovered in human urine. It has been isolated and identified by mass spectrometry and synthesis. The daily excretion in nine subjects on a free diet was 88 mumol/day (range, 32 to 144 mumol/day). cis-3,4-Methylene hexanedioic acid was given orally to a rat. About 90% of the dose was recovered unchanged in the urine within 24 h. Intragastric administration of cis-9,10-methylene [9,10-3H2]octadecanoic acid to rats gave four labeled urinary metabolites. The major one was cis-3,4-methylene hexanedioic acid, the others were 2,3-methylene pentanedioic acid and isomers of methylene heptanedioic acid and methylene octanedioic acid. Within 72 h, about 40% of the administered radioactivity could be recovered from the urine and another 40% from the carcass. About 20% of the recovered radioactivity was found to be water. Of the radioactivity administered to rats orally as cis-9,10-methylene [9,10-3H2]octadecanoic acid methyl ester, about 50% could be recovered from the lymph of the thoracic duct within 9 h. Intraperitoneal administration of cis-9,10-methylene octodecanoic acid methyl ester to rats gave the same metabolites. Of the given amount, 50 mol % could be recovered from the urine as cis-3,4-methylene hexanedioic acid and 19 mol % as homologues within 38 days.