Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.