In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.SdhR that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe–S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 μg l−1 carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 μg l−1 of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.SdhR cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0–30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60–95%.