This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe.