Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of Fusarium. As a consequence of infection with Fusarium culmorum and Fusarium graminearum, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator Saccharomyces cerevisiae strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 microg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic Fusarium mycotxins.