B-chromosomes (Bs) of two mammalian species, raccoon dog (Nyctereutesprocyonoides, Carnivora) and Asian wood mouse (Apodemus peninsulae, Rodentia) were investigated using chromosome segment microdissection and double-colour FISH. In the raccoon dog, all B-chromosomes showed homology with each other but not with the A-chromosomes. Two segment-specific probes (from proximal and distal parts of B) have been localized in corresponding chromosome parts, with significant variation in their sizes. In Asian wood mice, two types of B-specific chromatin were revealed--B1 and B2. Most Bs were either B1 or B2 specific; furthermore, some Bs were found to be composed of both types of chromatin. B-chromosome-specific libraries of A. peninsulae contain sequences homologous to the heterochromatic regions of sex and some A-chromosomes and dispersed repeated sequences. B1-specific probes gave signals on sex chromosomes of Apodemus speciosus and Apodemus agrarius. The origin and evolution of B-chromosomes in mammals are discussed.