The amino acid, dityrosine, is a major component of the spore wall surface of the yeast Saccharomyces cerevisiae, where it is part of a highly cross-linked macromolecular network of yet unknown chemical structure, consisting mostly of glucosamine, dityrosine and few other amino acids. Biosynthesis of the dityrosine moiety of this network consists of several steps, including the chemical modification of free L-tyrosine and the subsequent oxidative cross-linking of the modified tyrosine residues (catalyzed by a cytochrome P-450), leading to soluble dityrosine-containing spore wall precursors. We isolated, purified and characterized the dityrosine-containing precursor that appears late in spore wall synthesis and that is thought to be directly incorporated into the maturing spore wall. Chemical and spectroscopic analyses showed that this precursor is N,N'-bisformyl dityrosine. In addition, we identified a tyrosine-containing spore wall precursor as N-formyl tyrosine. The elucidation of the chemical structure of soluble spore wall precursors is crucial for the characterization of the function of the enzymes involved in maturation of the spore surface, e.g. by in vitro systems. A dityrosine-containing fragment, which was solubilized from mature spore walls by partial hydrolysis, was identified as N-formyl dityrosine. Mature spore walls contain significant amounts of N-formyl dityrosine and N,N'-bisformyl dityrosine. This supports the assumption that the dityrosine-containing macromolecular network on the spore surface has an unusual, non-peptidic structure.