During sporulation of Saccharomyces cerevisiae, the four haploid nuclei generated by meiosis are encapsulated within multilayered spore walls. Taking advantage of the natural fluorescence imparted to yeast spores by the presence of a dityrosine-containing macromolecule in the spore wall, we identified and cloned two genes, termed DIT1 and DIT2, which are required for spore wall maturation. Mutation of these genes has no effect on the efficiency of spore formation or spore viability. The mutant spores, however, fail to accumulate the spore wall-specific dityrosine and lack the outermost layer of the spore wall. The absence of this cross-linked surface layer reduces the resistance of the spores to lytic enzymes, to ether, and to elevated temperature. Expression of the DIT and DIT2 genes is restricted to sporulating cells, with the DIT1 transcripts accumulating at the time of prospore enclosure and just prior to the time of dityrosine biosynthesis. Both genes act in a spore-autonomous manner implying that at least some of the activities responsible for forming the outermost layer of the spore wall reside within the developing spore rather than in the surrounding ascal cytoplasm. As the DIT2 gene product has significant homology with cytochrome P-450s, DIT2 may be responsible for catalyzing the oxidation of tyrosine residues in the formation of dityrosine.