In the mouse, virtually every autosome pair, and the X and Y, was identified by its distinctive fluorescent banding pattern after staining with quinacrine mustard. The karyotype was characterized further by measurement of orcein-stained chromosomes and autoradiographic analysis of terminal DNA replication. The autoradiographic identification of the X and Y chromosomes was confirmed by this technique. Fluorescent karyotype analysis of inbred strains and a series of Fx hybrids involving C57BL/6J revealed a polymorphism, the presence or absence of a prominent secondary constriction on any of four different chromosomes, which was probably inherited in a simple Mendelian fashion.