Okadaic acid (1) is a polyether compound produced by the marine dinoflagellate Prorocentrum lima. Its biosynthesis attracts considerable attention since the carbon skeleton has been shown to be synthesized via an unusual route. However, a very limited amount of information is available for the formation of its ether rings. We applied collision-induced dissociation tandem mass spectrometry (CID MS/MS) to the elucidation of the O-18-incorporation pattern of okadaic acid. The extensive determination of O-18/O-16 ratios for each product ion bearing differing numbers of incorporated O-18 atoms resulted in the complete assignment of the labeled positions with accurate isotope ratios; the positions labeled from molecular oxygen (O-18(2)) were O(1)/O(2), O(3), O(5), O(6), O(8), O(9), O(10), and O(12). Those labeled from [O-18(2)]acetate were O(4), O(6), O(7), and O(11) (oxygen atoms are numbered beginning with those of carboxylic acid as O(1)/O(2) to O(13) in ring G). These incorporation patterns suggest that the cyclization of ether rings C, D, and E occurs via a beta-epoxide intermediate at C22-C23, and the carboxylic acid is formed by Baeyer-Villiger oxidation.