The de novo formation of multilayered spore walls inside a diploid mother cell is a major landmark of sporulation in the yeast Saccharomyces cerevisiae. Synthesis of the dityrosine-rich outer spore wall takes place toward the end of this process. Bisformyl dityrosine, the major building block of the spore surface, is synthesized in a multistep process in the cytoplasm of the prospores, transported to the maturing wall, and polymerized into a highly cross-linked macromolecule on the spore surface. Here we present evidence that the sporulation-specific protein Dtrlp (encoded by YBR180w) plays an important role in spore wall synthesis by facilitating the translocation of bisformyl dityrosine through the prospore membrane. DTR1 was identified in a genome-wide screen for spore wall mutants. The null mutant accumulates unusually large amounts of bisformyl dityrosine in the cytoplasm and fails to efficiently incorporate this precursor into the spore surface. As a result, many mutant spores have aberrant surface structures. Dtrlp, a member of the poorly characterized DHA12 (drug:H+ antiporter with 12 predicted membrane spans) family, is localized in the prospore membrane throughout spore maturation. Transport by Dtrlp may not be restricted to its natural substrate, bisformyl dityrosine. When expressed in vegetative cells, Dtrlp renders these cells slightly more resistant against unrelated toxic compounds, such as antimalarial drugs and food-grade organic acid preservatives. Dtrlp is the first multidrug resistance protein of the major facilitator superfamily with an assigned physiological role in the yeast cell.