We have devised an easy and effective genetic transformation method for the preeminent edible mushroom, Agaricus bisporus. Our method exploits the T-DNA transfer mechanism in Agrobacterium tumefaciens and relies on the reproductive fruiting body as the recipient tissue. The use of fruiting body explants, particularly the gill, provided high-frequency transformation, overcoming the inefficacy of Agrobacterium-based methods targeting fungal spores or vegetative mycelium. The protocol entails incubation of A. tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin. Between 7 and 28 d on the selection medium, upwards of 95% of the gill explants generate hygromycin-resistant colonies. About 75% of the mushroom transformants show a single-copy of the hygromycin-resistant gene integrated at random sites in the genome.