The chromosomes of the Chinese hamster established cell line CHEF-125 were studied by quinacrine fluorescence, acetic-saline-Giemsa staining, and a technique for localizing constitutive heterochromatin. The modal chromosome number was 22, but there were, in approximately equal proportions, two karyotypes with 22 chromosomes which differed by one chromosome only. Thirteen of the 22 chromosomes were normal ones and could be divided into five pairs of homologs and three in the haploid status. One chromosome was deficient for part of the short arm but otherwise normal, and one chromosome had an additional, unidentified segment. Of the seven new chromosomes, five were not identified, but two had sections of heterochromatin of the type and extent found in the sex chromosomes of the normal complement. One of these was composed in part of the long arm of the X chromosome.