The Roan locus is responsible for the coat coloration of Belgian Blue and Shorthorn cattle. The solid-colored and white animals are homozygotes, and the roan animals, with intermingled colored and white hairs, are heterozygous. The roan phenotype was mapped to cattle Chromosome (Chr) 5 with microsatellites, and a candidate gene was proposed (Charlier et al. Mamm Genome 7, 138, 1996). PCR primers to the exons of this candidate gene, the steel locus or mast cell growth factor (MGF) were designed. Solid-colored and white animals were sequenced. A missense mutation at 654 bp (amino acid 193, Ala ? Asp) was detected in these two groups. A PCR-RFLP was designed to this single base pair change, and 143 animals in total (Belgian Blue, Shorthorn, and various other breeds) were screened. In addition, the Canadian Beef Cattle Reference Herd (http://skyway.usask.ca/?schmutz) was used to verify Mendelian inheritance of this marker with the phenotypic inheritance of roan. Our data suggest that this mutation in the bovine MGF gene is responsible for the roan phenotype.