The major products of the initial steps of ferulic acid polymerization by lignin peroxidase included three dehydrodimers resulting from beta-5' and beta-beta'coupling and two trimers resulting from the addition of ferulic acid moieties to decarboxylated derivatives of beta-O-4'- and beta-5'-coupled dehydrodimers. This is the first time that trimers have been identified from peroxidase-catalyzed oxidation of ferulic acid, and their formation appears to be favored by decarboxylation of dehydrodimer intermediates. After initial oxidation, the coupling reactions appear to be determined by the chemistry of ferulic acid phenoxy radicals, regardless of the enzyme and of whether the reaction is performed in vitro or in vivo. This claim is supported by our finding that horseradish peroxidase provides a similar product profile. Furthermore, two of the dehydrodimers were the two products obtained from laccase-catalyzed oxidation (Tatsumi, K. S., Freyer, A., Minard, R. D., and Bollag, J.-M. (1994) Environ. Sci. Technol. 28, 210-215), and the most abundant dehydrodimer is the most prominent in grass cell walls (Ralph, J., Quideau, S., Grabber, J. H., and Hatfield, R. D. (1994) J. Chem. Soc. Perkin Trans. 1, 3485-3498). Our results also indicate that the dehydrodimers and trimers are further oxidized by lignin peroxidase, suggesting that they are only intermediates in the polymerization of ferulic acid. The extent of polymerization appears to be dependent on the ionization potential of formed intermediates, H(2)O(2) concentration, and, probably, enzyme stability.