Using fluorescence in situ hybridization (FISH), four different seed protein genes were physically mapped on metaphase chromosomes of Vicia faba L. dropped on slides. FISH with a 2.8 kb genomic probe of a legumin B4 gene resulted in reproducible signals on the long arm of chromosome III near the centromere. The same clone cross-hybridized at a lower frequency to the short arm of chromosome II, where the closely related legumin B3 gene family is located. The locus for legumin A-genes could be detected in the distal half of the long arm of chromosome V using a 1.7 kb cDNA clone. The locus of an unknown seed protein gene was mapped to the long arm of chromosome I using a mixture of polymerase chain reaction-amplified DNA fragments of the coding region of up to 1 kb in size.