Chromosome preparations from three sublines of mouse L-cells (A9, B82 L60) were made by conventional air-drying methods and the slides treated both by trypsin digestion and the C-banding methods, in order to investigate and compare the distribution of constitutive heterochromatin in these cells. Comparisons were also made with human and Chinese hamster cells. The mouse heterochromatic sites observed, including the interstitial sites found on the marker chromosomes, were similar for each line irrespective of the method used. The interstitial heterochromatic sites in the marker chromosomes 1 and 3 correspond to the locations of the secondary constrictions. The trypsin digestion method reveals the sites of constitutive heterochromatin in mouse chromosomes, but not in human or Chinese hamster chromosomes. All mouse chromosomes could be distinguished from those of Chinese hamster in the hybrid cells between mouse and Chinese hamster cell lines. The method is simple to use and therefore will facilitate the identification of mouse chromosomes within and between the cell populations of different origins and within cell hybrids.