Besides the well-known applications of bacterial artificial chromosomes (BACs) in classical molecular genetics, BACs are also used for molecular cytogenetic studies. BACs, as well as other locus-specific probes like cDNA, plasmids, cosmids, fosmids, P1-clones or yeast artificial chromosomes (YACs), can be labeled with fluorochromes and applied in FISH experiments. Various applications are possible, like gene mapping, FISH banding, determination of chromosomal breakpoints, characterization of derivative chromosomes, studies of interphase architecture, and karyotypic evolution studies. Here the basic principle of hybridizing BACs in situ on chromosome preparations is outlined. Moreover, an overview of possible issues that can be studied using BACs as FISH probes is provided. Finally, a shortened and more efficient FISH protocol using microwave treatment which yields results that can be evaluated within a few hours is presented.